Benzimidazole Inhibitors of the Sodium Channel

ABSTRACT

The invention relates to compounds useful in treating conditions associated with voltage-gated ion channel function, particularly conditions associated with sodium channel activity. More specifically, the invention concerns compounds (e.g., compounds according to any of Formulas (I)-(XIII) or Compounds (1)-(236) of Table 1) that are useful in treatment of a variety of diseases and conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Application Nos.61/448,923 and 61/448,910, each of which was filed on Mar. 3, 2011, andeach of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to compounds useful in treating conditionsassociated with voltage-gated ion channel function, particularlyconditions associated with sodium channel activity. More specifically,the invention concerns benzimidazole compounds that are useful intreatment numerous diseases and conditions.

BACKGROUND OF THE INVENTION

Voltage-gated sodium (Na_(v)) channels are present in neurons andexcitable tissues where they contribute to processes such as membraneexcitability and muscle contraction (Ogata et al., Jpn. J. Pharmacol.(2002) 88(4) 365-77). Nine different transmembrane α-subunits(Na_(v)1.1-1.9) from a single Na_(v)1 family combine with auxiliaryβ-subunits that modify channel function to form functional Na_(v)channels. Of the nine Na_(v)1 α-subunit isoforms, five are expressed inthe dorsal root ganglion where they are involved in setting the restingmembrane potential and the threshold for generating action potentials,and also contribute to the upstroke as well as firing of actionpotentials during sustained depolarization. In particular, thetetrodotoxin (TTX) sensitive Na_(v)1.7 and TTX-insensitive Na_(v)1.8channel subtypes act as major contributors to both inflammatory andneuropathic pain (Momin et al., Curr Opin Neurobiol. 18(4):383-8, 2008;Rush et al., J Physiol. 579(Pt 1):1-14, 2007).

Novel allosteric modulators of voltage-gated ion channels (e.g., sodiumchannels) are thus desired. Modulators may affect the kinetics and/orthe voltage potentials of, e.g., Na_(v)1.7 and/or Na_(v)1.8 channels.

SUMMARY OF THE INVENTION

The invention relates to compounds useful in conditions modulated byvoltage-gated ion channels (e.g., voltage gated sodium channels).

In a first aspect, the invention features a compound having a structureaccording to the following formula,

or a pharmaceutically acceptable salt or solvate thereof, where

each of R¹, R², R³, and R⁴ is selected, independently, from H,optionally substituted C1-C6 alkyl, optionally substituted C1-C6haloalkyl, optionally substituted C6-C10 aryl, or optionally substituted5 to 6-membered heteroaryl, where at least one of R¹, R², R³, and R⁴ ishalogen or optionally substituted C1-C6 haloalkyl;

R⁵ is H, optionally substituted C1-C6 alkyl, or optionally substitutedC1-C10 heteroalkyl;

R⁶ is —R^(6A) or —CH₂R^(6B);

R^(6A) is NH₂, optionally substituted cyclopropyl, optionallysubstituted azetidine, optionally substituted cyclopentyl, optionallysubstituted pyrazole, optionally substituted pyrrole, optionallysubstituted pyrrolidine, optionally substituted thiazolidine, optionallysubstituted thiazolidine-1,1-dioxide, optionally substituted pyrimidine,optionally substituted C1-C10 aminoalkyl, optionally substituted C1-C10hydroxyalkyl, optionally substituted C1-C10 alkoxyalkyl, optionallysubstituted C1-C10 haloalkyl, or optionally substituted C1-C10alkylsulfonyl; or R^(6A) has a structure according to

where

n is an integer between 0-4;

Z¹ is CH₂, NH, NCH₃, or O;

L¹ is —CH₂, —CHR^(4A), —CH₂C(═O), —C(═O)CH₂, —CH₂C(═O)NH,—CH₂C(═O)NHCH₂, or —CH₂NHC(═O)CH₂;

each R^(2A) and R^(2C), when present, is selected from OH, N(R^(2B))₂,halogen, and unsubstituted C1-C3 alkyl, or two R² combine to form an oxo(═O) group, and wherein no more than two R^(2A) combine to form an oxogroup; and

each R^(2B) is, independently, H or unsubstituted C1-C6 alkyl;

R^(2D) is H, OH, or NH₂;

and

R^(6B) is optionally substituted cyclopropyl, optionally substitutedazetidine, optionally substituted cyclopentyl, optionally substitutedpyrazole, optionally substituted pyrrole, optionally substitutedpyrrolidine, optionally substituted thiazolidine, optionally substitutedthiazolidine-1,1-dioxide, or optionally substituted pyrimidine.

In some embodiments, R^(6A) is NH₂, optionally substituted cyclopropyl,optionally substituted azetidine, optionally substituted cyclopentyl,optionally substituted pyrazole, optionally substituted pyrrole,optionally substituted pyrrolidine, optionally substituted thiazolidine,optionally substituted thiazolidine-1,1-dioxide, optionally substitutedpyrimidine, optionally substituted C1-C10 aminoalkyl, optionallysubstituted C1-C10 hydroxyalkyl, optionally substituted C1-C10alkoxyalkyl, optionally substituted C1-C10 haloalkyl, or optionallysubstituted C1-C10 alkylsulfonyl.

In other embodiments, R⁶ has a structure according to

In some embodiments, R⁵ is H.

In other embodiments, R⁵ is optionally substituted C1-C10 heteroalkyl.

In certain embodiments, R² and R⁴ are both CF₃, F, or Cl.

In still other embodiments, R² and R³ are both CF₃, F, or Cl.

In some embodiments, R⁶ is —CH₂R^(6B), and R^(6B) is optionallysubstituted azetidine.

In certain embodiments, R⁶ is optionally substituted C1-C10 aminoalkyl.

In other embodiments, the C1-C10 aminoalkyl includes an oxo (═O)substituent, an alkoxy substituent, an N-sulfonyl group, or anycombination thereof.

In some embodiments, the compound has a structure according to thefollowing formula.

or a pharmaceutically acceptable salt or solvate thereof, where n is 0or 1, and R⁷ is H or —C(═O)R^(7A), where R^(7A) is unsubstituted C1-C6alkyl or optionally substituted C1-C10 aminoalkyl.

In certain embodiments, n is 0.

In other embodiments, n is 1.

In some embodiments, R⁷ is H or C(═O)R^(7A), where R^(7A) isunsubstituted C1-C3 alkyl or an optionally substituted C1-C10 aminoalkylincluding a terminal —NH₂ group.

In further embodiments, R² and R⁴ are both CF₃, F, or Cl.

In some embodiments, R² and R³ are both CF₃, F, or Cl.

In other embodiments, R⁶ is optionally substituted cyclopropyl,optionally substituted azetidine, optionally substituted cyclopentyl,optionally substituted pyrazole, optionally substituted pyrrole,optionally substituted pyrrolidine, optionally substituted thiazolidine,optionally substituted thiazolidine-1,1-dioxide, optionally substitutedpyrimidine.

In still other embodiments, R⁶ includes a —NH₂ substituent.

In some embodiments, the compound has a structure according to thefollowing formula,

where R^(6B) is optionally substituted azetidine, optionally substitutedcyclopentyl, optionally substituted pyrrolidine, optionally substitutedthiazolidine, optionally substituted thiazolidine-1,1-dioxide, oroptionally substituted pyrimidine.

In certain embodiments, R² and R⁴ are both CF₃, F, or Cl.

In other embodiments, R² and R³ are both CF₃, F, or Cl.

In further embodiments, R⁵ is H.

In still other embodiments, R⁶ is an optionally substituted C1-C10aminoalkyl group.

In some embodiments, the C1-C10 aminoalkyl includes a terminal —NH₂group.

In other embodiments, the C1-C10 aminoalkyl includes an oxo (═O)substituent.

In certain embodiments, R⁶ is—(CH₂)_(m1)(NR^(6C))_(m2)(C═O)_(m3)(CH₂)_(m4)NR^(6D)R^(6E) or—(CH₂)_(m1)(C(CH₃)₂)_(m2)(CH₂)_(m4)NR^(6C)R^(6D) where each of m1 and m4is, independently, an integer between 1-6; each of m2 and m3 is,independently, 0 or 1; each of R^(6C) and R^(6E) is, independently, H orunsubstituted C1-C6 alkyl; and R^(6D) is H, unsubstituted C1-C6 alkyl,or an N-protecting group.

In some embodiments, R⁶ is —(CH₂)_(m1)NH₂, —CH₂NHC(═O)CH₂NH₂,—C(CH₃)₂CH₂NH₂, —C(CH₃)₂NH₂, and where m1 is 1, 2, or 3.

In other embodiments, R² and R⁴ are both CF₃, F, or Cl.

In still other embodiments, R² and R³ are both CF₃, F, or Cl.

In particular embodiments, one and only one of R² or R³ is optionallysubstituted phenyl.

In certain embodiments, R⁵ is H.

In some embodiments, R⁶ is optionally substituted C1-C3 haloalkyl,optionally substituted C1-C10 alkoxyalkyl, optionally substituted C1-C10hydroxyalkyl, or optionally substituted C1-C10 alkylsulfonyl.

In other embodiments, R⁶ is —(CH₂)_(m1)CF₃, —(CH₂)_(m1)OR^(6F),—(CH₂)_(m1)SO₂R^(6G), where m1 is an integer between 1-6, R^(6F) is H orCH₃, and R^(6G) is unsubstituted C1-C3 alkyl.

In further embodiments, R² and R⁴ are both CF₃, F, or Cl.

In certain embodiments, R² and R³ are both CF₃, F, or Cl.

In some embodiments, R⁵ is H.

In a second aspect, the invention features a compound having a structureaccording to the following formula,

or a pharmaceutically acceptable salt or solvate thereof, where

each of X¹, X², and X³ is N or CR⁴, and where one and only one of X¹,X², and X³ is N;

L¹ is a covalent bond, —CH₂, —CHR^(5A), —CH₂C(═O), —C(═O)CH₂,—CH₂C(═O)NH, —CH₂C(═O)NHCH₂, —CH₂NHC(═O)CH₂, or —CH₂CH₂;

each of R¹, R², and R⁴ is, independently, H, unsubstituted C1-C3 alkyl,optionally substituted C1-C3 haloalkyl, or halogen;

R³ is H, optionally substituted C1-C6 alkyl, or optionally substitutedC1-C10 heteroalkyl; R^(5A) is selected from optionally substituted C1-C3alkyl; and

where at least one of R¹, R², R³, and R⁵ is halogen or optionallysubstituted C1-C3 haloalkyl.

In some embodiments, L¹ is CH₂ or CHCF₃.

In other embodiments, R³ is H.

In another aspect, the invention features a compound having a structureaccording to the following formula,

or a pharmaceutically acceptable salt or solvate thereof, where

n is an integer between 0-4;

R¹ is selected from —CH₂R^(3A), —CHR^(4A)R^(3A), —CH₂C(═O)R^(3A),—C(═O)CH₂R^(3A), —CH₂C(═O)NR^(4B)R^(3A), CH₂C(═O)NR^(4B)CH₂R^(3A),—CH₂NR^(4B)C(═O)CH₂R^(3A), —R^(3B), —CH₂CH₂R^(3B), and—CH₂C(═O)NR^(4B)CHR^(4C)R^(3C);

each R², when present, is selected from OH, N(R^(2A))₂, halogen, andunsubstituted C1-C3 alkyl, or two R² combine to form an oxo (═O) group,and where no more than two R² combine to form an oxo group;

-   -   each R^(2A) is, independently, H or unsubstituted C1-C6 alkyl;

R^(3A) is selected from

-   -   a benzimidazole including at least one C-substituent selected        from halogen or C1-C6 haloalkyl or an N-substituent that is        C1-C12 heteroalkyl;    -   a pyridine including at least one substituent selected from        halogen or C1-C6 haloalkyl; and    -   a pyrazole including at least one substituent selected from        halogen, unsubstituted C1-C3 alkyl, and C1-C6 haloalkyl;

R^(3B) is selected from

-   -   a benzimidazole including at least one C-substituent selected        from Cl, Br, I, or C1-C6 haloalkyl or an N-substituent that is a        C1-C10 heteroalkyl;    -   a pyridine including at least one substituent selected from        halogen or C1-C6 haloalkyl; and    -   a pyrazole including at least one substituent selected from        halogen, unsubstituted C1-C3 alkyl, and C1-C6 haloalkyl;

R^(3C) is optionally substituted pyridine;

R^(4A) is optionally substituted C1-C3 alkyl;

R^(4B) is H or optionally substituted C1-C3 alkyl;

R^(4C) is C1-C3 haloalkyl;

Z¹ is selected from CH₂, O, and NR⁵, where R⁵ is H or unsubstitutedC1-C6 alkyl; and

Z² is NH, NR⁶, CHR², CR⁶R², where R⁶ is a covalent bond to R¹.

In some embodiments, n is 0.

In other embodiments, n is 2 or 4. In further embodiments, two R²combine to form an oxo group. In certain embodiments, R² is CH₃.

In some embodiments, Z¹ is O, NH, CH₂, or NCH₃.

In other embodiments, Z² is N, CH, or CNH₂.

The compounds described herein can have a structure according to thefollowing formula,

or a pharmaceutically acceptable salt thereof, where

Z¹ is CH₂, NH, NCH₃, or O;

L¹ is —CH₂, —CHR^(4A), —CH₂C(═O), —C(═O)CH₂, —CH₂C(═O)NH,—CH₂C(═O)NHCH₂, or —CH₂NHC(═O)CH₂;

R⁵ is H or C1-C10 heteroalkyl;

each of R¹, R², R³, and R⁴ is, independently, H, unsubstituted C1-C3alkyl, optionally substituted C1-C3 haloalkyl, or halogen, and

where at least one of R¹, R², R³, R⁴, and R^(R5) is not H.

In some embodiments, Z¹ is NH.

In other embodiments, n is 2 or 4.

In some embodiments, two R^(2A) combine to form an oxo group.

In other embodiments, R^(2A) is CH₃.

In still other embodiments, R¹ and R⁴ are both H.

In certain embodiments, R² and R⁴ are, independently, F, CF₃, or Cl.

In some embodiments, R³ is F, Cl, or CF₃.

In other embodiments, R² is F, Cl, or CF₃.

In still other embodiments, R⁵ is H.

In further embodiments, R⁵ is optionally substituted C1-C10 hydroxyalkylor C1-C10 aminoalkyl.

In other embodiments, L¹ is CH₂.

In other embodiments, the compound has a structure according to thefollowing formula,

or a pharmaceutically acceptable salt thereof, where

Z¹ is CH₂, NH, NCH₃, or O;

each of X¹, X², and X³ is N or CR^(8C), and where one and only one ofX¹, X², and X³ is N;

L¹ is a covalent bond, —CH₂, —CHR^(4A), —CH₂C(═O), —C(═O)CH₂,—CH₂C(═O)NH, —CH₂C(═O)NHCH₂, —CH₂NHC(═O)CH₂, or —CH₂CH₂;

each of R^(8A), R^(8B), and R^(8C) is, independently, H, unsubstitutedC1-C3 alkyl, optionally substituted C1-C3 haloalkyl, or halogen, and

where at least one of R⁷, R^(8A), R^(8B), R^(8C), and R^(8D) is not H.

In some embodiments, Z¹ is NH.

In other embodiments, n is 2 or 4. In certain embodiments, two R²combine to form an oxo group.

In further embodiments, R² is CH₃.

In some embodiments, X² is N.

In still other embodiments, at least one of R^(8A), R^(8B), and R^(8C)is F, Cl, or CF₃.

In some embodiments, L¹ is —CH₂C(═O)NHCH₂ or —CH₂NHC(═O)CH₂.

In other embodiments, the compound has a structure according to thefollowing formula,

or a pharmaceutically acceptable salt or solvate thereof wherein

Z¹ is CH₂ or NH;

L¹ is a covalent bond, —CH₂, —CHR^(4A), —CH₂C(═O), —C(═O)CH₂,—CH₂C(═O)NH, —CH₂C(═O)NHCH₂, —CH₂C(═O)NHCHCF₃—, or —CH₂NHC(═O)CH₂;

n is an integer between 0-4;

each R^(2C), when present, is independently, OH, NH₂, NHCH₃, N(CH₃)₂, orunsubstituted C1-C3 alkyl, or two R^(2C) groups combine to form an oxo(═O) group, and wherein no more than one R^(2B) or R^(2C) group can beOH NH₂, NHCH₃, or N(CH₃)₂;

R^(2D) is H, OH, or NH₂,

R⁵ is H or C1-C10 heteroalkyl;

each of R¹, R², R³, and R⁴ is, independently, H, unsubstituted C1-C3alkyl, optionally substituted C1-C3 haloalkyl, or halogen, and

wherein at least one of R¹, R², R³, R⁴, and R⁵ is not H.

In certain embodiments, Z¹ is CH₂.

In other embodiments, n is 0 or 1.

In certain embodiments, one of R² and R³ is NH₂, NHCH₃, or N(CH₃)₂.

In further embodiments, R¹ and R⁴ are both H.

In other embodiments, R¹ and R⁴ are, independently, F, CF₃, or Cl.

In still other embodiments, R³ is F, Cl, or CF₃.

In certain embodiments, R² is F, Cl, or CF₃.

In some embodiments, R⁵ is H.

In certain embodiments, the compound has a structure according to thefollowing formula,

or a pharmaceutically acceptable salt or solvate thereof, where

Z¹ is CH₂, NH, NCH₃, or O;

L¹ is a covalent bond, —CH₂, —CHR^(4A), —CH₂C(═O), —C(═O)CH₂,—CH₂C(═O)NH, —CH₂C(═O)NHCH₂, —CH₂NHC(═O)CH₂, or —CH₂CH₂;

R⁷ is selected from H, optionally substituted C1-C6 alkyl, andoptionally substituted C1-C10 heteroalkyl;

each of R^(8A) and R^(8B) is selected, independently, from H, halogen,unsubstituted C1-C3 alkyl, and C1-C3 haloalkyl, and

where at least one of R⁷, R^(8A), and R^(8B) is not H.

In some embodiments, Z¹ is N.

In other embodiments, n is 2 or 4.

In still other embodiments, two R² combine to form an oxo group.

In certain embodiments, R² is CH₃.

In some embodiments, R⁷ is unsubstituted C1-C3 alkyl.

In further embodiments, at least one of R^(8A) and R^(8B) is F, Cl, orCF₃.

In some embodiments, L¹ is CH₂.

In still another aspect, the invention features a compound having astructure according to the following formula,

or a pharmaceutically acceptable salt or solvate thereof, where

each of R¹, R², and R³ is, independently, H, unsubstituted C1-C6 alkyl,optionally substituted C1-C6 haloalkyl, or halogen;

m is 1 or 2;

each R⁴ and R⁵ is, independently, H, optionally substituted C1-C6 alkyl,or optionally substituted C1-C6 haloalkyl, or R⁴ and R⁵ combine to forman optionally substituted C3-C6 cycloalkyl, or R⁴ and R⁵ combine to forman oxo (C═O) group;

each of R⁶ and R⁸ is, independently, H or optionally substituted C1-C6alkyl; or R⁶ and R⁸ combine to form an optionally substitutedthree-to-nine membered heterocyclyl, or R⁶ and R^(7A) combine to form anoptionally substituted three-to-nine membered heterocyclyl;

n is 1 or 2;

each R^(7A) and R^(7B) is, independently H, optionally substituted C1-C6alkyl, or optionally substituted C1-C6 haloalkyl; or R⁶ combines withR^(7A) to form an optionally substituted three-to-nine heterocyclyl; oran R^(7A) and R^(7B) group on the same carbon combine to form anoptionally substituted C3-C6 cycloalkyl; or, when n is 2, both R^(7A)groups combine to form an optionally substituted C3-C6 cycloalkyl.

In some embodiments, each of R¹, R², and R³ is, independently, H, C1-C3haloalkyl, or halogen.

In other embodiments, one of R¹, R², and R³ is H.

In still other embodiments, two of R¹, R², and R³ are, independently,CF₃, Cl, or F.

In certain embodiments, R⁴ and R⁵ are both H; or R⁴ and R⁵ are both CH₃;or R⁴ and R⁵ combine to form an optionally substituted C3-C6 cycloalkyl;or R⁴ and R⁵ combine to form an oxo (C═O) group.

In other embodiments, R⁶ combines with R^(7A) to form a three-to-sixmembered heterocyclyl ring, or wherein R⁶ and R⁸ combine to form anoptionally substituted three-to-six membered heterocyclyl.

In still other embodiments, R⁶ is H.

In some embodiments, R^(7A) is H and R^(7B) is optionally substitutedC1-C6 alkyl.

In still other embodiments, the compound has a structure according to aformula that is

In further embodiments, n is 1, and R^(7A) and R^(7B) are both H, orR^(7A) is H and R^(7B) is optionally substituted C1-C6 alkyl. In stillother embodiments, R⁶ is H and R⁸ is optionally substituted C1-C6 alkyl,or wherein R⁶ and R⁸ combine to form an optionally substituted five- tosix-membered heterocyclyl (e.g., an unsubstituted five- to six-memberedheterocyclyl or a five- to six-membered heterocyclyl that includes aphenyl substituent).

In another aspect, the invention features a compound having a structureselected from the group consisting of any of Compounds (1)-(236) ofTable 1, or a pharmaceutically acceptable salt or solvate thereof. Insome embodiments, the compound, or a pharmaceutically acceptable salt orsolvate thereof, selected from the group consisting of

In another aspect, the invention features a pharmaceutical compositionthat includes any of the compounds described herein (e.g., a compoundaccording to any of Formulas (I)-(XIII) or any of Compounds (1)-(236) ofTable 1) and a pharmaceutically acceptable carrier or excipient.

In some embodiments, the pharmaceutical composition is formulated inunit dosage form (e.g., a tablet, caplet, capsule, lozenge, film, strip,gelcap, or syrup).

In still another aspect, the invention features method to treat adisease or condition by administering to a subject in need of suchtreatment an effective amount of any of the compounds described herein(e.g., a compound according to any of Formulas (I)-(XIII) or any ofCompounds (1)-(236) of Table 1), or a pharmaceutical compositionthereof.

In certain embodiments, the disease or condition is pain, epilepsy,Parkinson's disease, a mood disorder (e.g., a major depressive disorder(e.g., atypical depression, melancholic depression, psychotic majordepression, catatonic depression, postpartum depression, seasonalaffective disorder, dysthymia, and depressive disorder not otherwisespecified (DD-NOS)), recurrent brief depression, minor depressivedisorder, or a bipolar disorder), psychosis (e.g., schizophrenia),tinnitus, amyotropic lateral sclerosis, glaucoma, ischaemia, spasticitydisorders, obsessive compulsive disorder, restless leg syndrome, andTourette syndrome.

In some embodiments, the subject is a fasted subject.

In certain embodiments, the subject is a fed subject.

In other embodiments, the condition is pain or epilepsy.

In some embodiments, the pain is inflammatory pain (e.g., inflammatorypain caused by rheumatoid arthritis, juvenile idiopathic arthritis,ankylosing spondylitis, psoriatic arthritis, inflammatory bowel disease,primary dysmenorrhea, or endometriosis) or neuropathic pain.

In certain embodiments, the pain is chronic pain.

In further embodiments, the chronic pain is peripheral neuropathic pain;central neuropathic pain, musculoskeletal pain, headache, visceral pain,or mixed pain.

In some embodiments, the peripheral neuropathic pain is post-herpeticneuralgia, diabetic neuropathic pain, neuropathic cancer pain,HIV-associated neuropathy, erythromelalgia, failed back-surgerysyndrome, trigeminal neuralgia, or phantom limb pain; said centralneuropathic pain is multiple sclerosis related pain, Parkinson diseaserelated pain, post-stroke pain, post-traumatic spinal cord injury pain,lumbosacral radiculopathy, cervical radiculopathy, brachialradiculopathy, or pain in dementia; the musculoskeletal pain isosteoarthritic pain and fibromyalgia syndrome; inflammatory pain such asrheumatoid arthritis, or endometriosis; the headache is migraine,cluster headache, tension headache syndrome, facial pain, or headachecaused by other diseases; the visceral pain is interstitial cystitis,irritable bowel syndrome, or chronic pelvic pain syndrome; or the mixedpain is lower back pain, neck and shoulder pain, burning mouth syndrome,or complex regional pain syndrome.

In other embodiments, the headache is migraine.

In certain embodiments, the pain is acute pain.

In further embodiments, the acute pain is nociceptive pain orpost-operative pain.

In another aspect, the invention features a method of modulating avoltage-gated sodium channel, the method including contacting a cellwith any of the compounds described herein (e.g., a compound accordingto any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1).

The term “alkoxy” represents a chemical substituent of formula —OR,where R is an optionally substituted C1-C6 alkyl group, unless otherwisespecified. In some embodiments, the alkyl group can be substituted,e.g., the alkoxy group can have 1, 2, 3, 4, 5 or 6 substituent groups asdefined herein.

The term “alkoxyalkyl” represents a heteroalkyl group, as definedherein, that is described as an alkyl group that is substituted with analkoxy group. Exemplary unsubstituted alkoxyalkyl groups include between2 to 12 carbons. In some embodiments, the alkyl and the alkoxy each canbe further substituted with 1, 2, 3, or 4 substituent groups as definedherein for the respective group.

As used herein, the term “alkyl,” “alkenyl” and “alkynyl” includestraight-chain, branched-chain and cyclic monovalent substituents, aswell as combinations of these, containing only C and H whenunsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl,cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The term“cycloalkyl,” as used herein, represents a monovalent saturated orunsaturated non-aromatic cyclic alkyl group having between three to ninecarbons (e.g., a C3-C9 cycloalkyl), unless otherwise specified, and isexemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, bicyclo[2.2.1.]heptyl, and the like. When the cycloalkylgroup includes one carbon-carbon double bond, the cycloalkyl group canbe referred to as a “cycloalkenyl” group. Exemplary cycloalkenyl groupsinclude cyclopentenyl, cyclohexenyl, and the like.

Typically, the alkyl, alkenyl and alkynyl groups contain 1-12 carbons(e.g., C1-C12 alkyl) or 2-12 carbons (e.g., C2-C12 alkenyl or C2-C12alkynyl). In some embodiments, the alkyl groups are C1-C8, C1-C6, C1-C4,C1-C3, or C1-C2 alkyl groups; or C2-C8, C2-C6, C2-C4, or C2-C3 alkenylor alkynyl groups. Further, any hydrogen atom on one of these groups canbe replaced with a substituent as described herein.

Heteroalkyl, heteroalkenyl and heteroalkynyl are similarly defined andcontain at least one carbon atom but also contain one or more O, S or Nheteroatoms or combinations thereof within the backbone residue wherebyeach heteroatom in the heteroalkyl, heteroalkenyl or heteroalkynyl groupreplaces one carbon atom of the alkyl, alkenyl or alkynyl group to whichthe heteroform corresponds. In some embodiments, the heteroalkyl,heteroalkenyl and heteroalkynyl groups have C at each terminus to whichthe group is attached to other groups, and the heteroatom(s) present arenot located at a terminal position. As is understood in the art, theseheteroforms do not contain more than three contiguous heteroatoms. Insome embodiments, the heteroatom is O or N. The term “heterocyclyl,” asused herein represents cyclic heteroalkyl or heteroalkenyl that is,e.g., a 3-, 4-, 5-, 6- or 7-membered ring, unless otherwise specified,containing one, two, three, or four heteroatoms independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur. The5-membered ring has zero to two double bonds, and the 6- and 7-memberedrings have zero to three double bonds. The term “heterocyclyl” alsorepresents a heterocyclic compound having a bridged multicyclicstructure in which one or more carbons and/or heteroatoms bridges twonon-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group.The term “heterocyclyl” includes bicyclic, tricyclic, and tetracyclicgroups in which any of the above heterocyclic rings is fused to one,two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring,a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or anothermonocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl,tetrahydroquinolyl, benzofuryl, benzothienyl and the like.

The designated number of carbons in heteroforms of alkyl, alkenyl andalkynyl includes the heteroatom count. For example, if heteroalkyl isdefined as C1-C6, it will contain 1-6 C, N, O, or S atoms such that theheteroalkyl contains at least one C atom and at least one heteroatom,for example 1-5 carbons and 1 N atom, or 1-4 carbons and 2 N atoms.Similarly, when heteroalkyl is defined as C1-C6 or C1-C4, it wouldcontain 1-5 carbons or 1-3 carbons respectively, i.e., at least one C isreplaced by O, N or S. Accordingly, when heteroalkenyl or heteroalkynylis defined as C2-C6 (or C2-C4), it would contain 2-6 or 2-4 C, N, O, orS atoms, since the heteroalkenyl or heteroalkynyl contains at least onecarbon atom and at least one heteroatom, e.g. 2-5 carbons and 1 N atom,or 2-4 carbons, and 2 O atoms. Further, heteroalkyl, heteroalkenyl orheteroalkynyl substituents may also contain one or more carbonyl groups.Examples of heteroalkyl, heteroalkenyl and heteroalkynyl groups includeCH₂OCH₃, CH₂N(CH₃)₂, CH₂OH, (CH₂)_(n)NR₂, OR, COOR, CONR₂, (CH₂)_(n)OR,(CH₂)_(n)COR, (CH₂)_(n)COOR, (CH₂)SR, (CH₂)_(n)SOR, (CH₂)_(n)SO₂R,(CH₂)_(n)CONR₂, NRCOR, NRCOOR, OCONR₂, OCOR and the like wherein the Rgroup contains at least one C and the size of the substituent isconsistent with the definition of e.g., alkyl, alkenyl, and alkynyl, asdescribed herein (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12).

As used herein, the terms “alkylene,” “alkenylene,” and “alkynylene,” orthe prefix “alk” refer to divalent or trivalent groups having aspecified size, typically C1-C2, C1-C3, C1-C4, C1-C6, or C1-C8 for thesaturated groups (e.g., alkylene or alk) and C2-C3, C2-C4, C2-C6, orC2-C8 for the unsaturated groups (e.g., alkenylene or alkynylene). Theyinclude straight-chain, branched-chain and cyclic forms as well ascombinations of these, containing only C and H when unsubstituted.Because they are divalent, they can link together two parts of amolecule, as exemplified by X in the compounds described herein.Examples are methylene, ethylene, propylene, cyclopropan-1,1-diyl,ethylidene, 2-butene-1,4-diyl, and the like. These groups can besubstituted by the groups typically suitable as substituents for alkyl,alkenyl and alkynyl groups as set forth herein. Thus C═O is a C1alkylene that is substituted by ═O, for example. For example, the term“alkaryl,” as used herein, represents an aryl group, as defined herein,attached to the parent molecular group through an alkylene group, asdefined herein, and the term “alkheteroaryl” refers to a heteroarylgroup, as defined herein, attached to the parent molecular group throughan alkylene group, as defined herein. The alkylene and the aryl orheteroaryl group are each optionally substituted as described herein.

Heteroalkylene, heteroalkenylene and heteroalkynylene are similarlydefined as divalent groups having a specified size, typically C1-C3,C1-C4, C1-C6, or C1-C8 for the saturated groups and C2-C3, C2-C4, C2-C6,or C2-C8 for the unsaturated groups. They include straight chain,branched chain and cyclic groups as well as combinations of these, andthey further contain at least one carbon atom but also contain one ormore O, S or N heteroatoms or combinations thereof within the backboneresidue, whereby each heteroatom in the heteroalkylene, heteroalkenyleneor heteroalkynylene group replaces one carbon atom of the alkylene,alkenylene or alkynylene group to which the heteroform corresponds. Asis understood in the art, these heteroforms do not contain more thanthree contiguous heteroatoms.

The term “alkylsulfonyl,” as used herein, represents a heteroalkyl groupthat is described as an optionally substituted alkyl group, as describedherein, that includes an —S(O)₂— group.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein eachR^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2),SOR^(N2), SO₂N(R^(N2))₂, SON(R^(N2))₂, an N-protecting group, alkyl,alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl,heterocyclyl (e.g., heteroaryl), alkheterocyclyl (e.g., alkheteroaryl),or two R^(N1) combine to form a heterocyclyl or an N-protecting group,and wherein each R^(N2) is, independently, H, alkyl, or aryl. In apreferred embodiment, amino is —NH₂, or —NHR^(N1), wherein R^(N1) is,independently, OH, NO₂, NH₂, NR^(N2), SO₂OR^(N2), SO₂R^(N2), SOR^(N2),SO₂N(R^(N2))₂, SON(R^(N2))₂, alkyl, or aryl, and each R^(N2) can be H,alkyl, or aryl. The term “aminoalkyl,” as used herein, represents aheteroalkyl group, as defined herein, that is described as an alkylgroup, as defined herein, substituted by an amino group, as definedherein. The alkyl and amino each can be further substituted with 1, 2,3, or 4 substituent groups as described herein for the respective group.For example, the alkyl moiety may comprise an oxo (═O) substituent.

“Aromatic” moiety or “aryl” moiety refers to any monocyclic or fusedring bicyclic system which has the characteristics of aromaticity interms of electron distribution throughout the ring system and includes amonocyclic or fused bicyclic moiety such as phenyl or naphthyl;“heteroaromatic” or “heteroaryl” also refers to such monocyclic or fusedbicyclic ring systems containing one or more heteroatoms selected fromO, S and N. The inclusion of a heteroatom permits inclusion of5-membered rings to be considered aromatic as well as 6-membered rings.Thus, typical aromatic/heteroaromatic systems include pyridyl,pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl,quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl,thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl, benzoisoxazolyl,imidazolyl and the like. Because tautomers are theoretically possible,phthalimido is also considered aromatic. Typically, the ring systemscontain 5-12 ring member atoms or 6-10 ring member atoms. In someembodiments, the aromatic or heteroaromatic moiety is a 6-memberedaromatic rings system optionally containing 1-2 nitrogen atoms. Moreparticularly, the moiety is an optionally substituted phenyl, pyridyl,indolyl, pyrimidyl, pyridazinyl, benzothiazolyl or benzimidazolyl,pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, benzothiazolyl, indolyl.Even more particularly, such moiety is phenyl, pyridyl, or pyrimidyl andeven more particularly, it is phenyl.

“O-aryl” or “O-heteroaryl” refers to aromatic or heteroaromatic systemswhich are coupled to another residue through an oxygen atom. A typicalexample of an O-aryl is phenoxy. Similarly, “arylalkyl” refers toaromatic and heteroaromatic systems which are coupled to another residuethrough a carbon chain, saturated or unsaturated, typically of C1-C8,C1-C6, or more particularly C1-C4 or C1-C3 when saturated or C2-C8,C2-C6, C2-C4, or C2-C3 when unsaturated, including the heteroformsthereof. For greater certainty, arylalkyl thus includes an aryl orheteroaryl group as defined above connected to an alkyl, heteroalkyl,alkenyl, heteroalkenyl, alkynyl or heteroalkynyl moiety also as definedabove. Typical arylalkyls would be an aryl(C6-C12)alkyl(C1-C8),aryl(C6-C12)alkenyl(C2-C8), or aryl(C6-C12)alkynyl(C2-C8), plus theheteroforms. A typical example is phenylmethyl, commonly referred to asbenzyl.

Halo may be any halogen atom, especially F, Cl, Br, or I, and moreparticularly it is fluoro or chloro.

The term “haloalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by a halogen group (i.e., F, Cl, Br, or I).A haloalkyl may be substituted with one, two, three, or, in the case ofalkyl groups of two carbons or more, four halogens. Haloalkyl groupsinclude perfluoroalkyls. In some embodiments, the haloalkyl group can befurther substituted with 1, 2, 3, or 4 substituent groups as describedherein for alkyl groups.

The term “hydroxy,” as used herein, represents an —OH group.

The term “hydroxyalkyl,” as used herein, represents an alkyl group, asdefined herein, substituted by one to three hydroxy groups, with theproviso that no more than one hydroxy group may be attached to a singlecarbon atom of the alkyl group, and is exemplified by hydroxymethyl,dihydroxypropyl, and the like.

The term “N-protecting group,” as used herein, represents those groupsintended to protect an amino group against undesirable reactions duringsynthetic procedures. Commonly used N-protecting groups are disclosed inGreene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (JohnWiley & Sons, New York, 1999), which is incorporated herein byreference. N-protecting groups include acyl, aryloyl, or carbamyl groupssuch as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl,2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl,phthalyl, o-nitropbenoxyacetyl, α-chlorobutyryl, benzoyl,4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliariessuch as protected or unprotected D, L or D, L-amino acids such asalanine, leucine, phenylalanine, and the like; sulfonyl-containinggroups such as benzenesulfonyl, p-toluenesulfonyl, and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyl oxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl,t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl,ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl 4-nitrophenoxy carbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and thelike, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl,and the like and silyl groups such as trimethylsilyl, and the like.Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl,t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc),and benzyloxycarbonyl (Cbz).

In general, a substituent group (e.g., alkyl, alkenyl, alkynyl, or aryl(including all heteroforms defined above) may itself optionally besubstituted by additional substituents. The nature of these substituentsis similar to those recited with regard to the substituents on the basicstructures above. Thus, where an embodiment of a substituent is alkyl,this alkyl may optionally be substituted by the remaining substituentslisted as substituents where this makes chemical sense, and where thisdoes not undermine the size limit of alkyl per se; e.g., alkylsubstituted by alkyl or by alkenyl would simply extend the upper limitof carbon atoms for these embodiments, and is not included. For example,where a group is substituted, the group may be substituted with 1, 2, 3,4, 5, or 6 substituents. Optional substituents include, but are notlimited to: C1-C6 alkyl or heteroaryl, C2-C6 alkenyl or heteroalkenyl,C2-C6 alkynyl or heteroalkynyl, halogen; aryl, heteroaryl, azido (—N₃),nitro (—NO₂), cyano (—CN), acyloxy (—OC(═O)R′), acyl (—C(═O)R′), alkoxy(—OR′), amido (—NR′C(═O)R″ or —C(═O)NRR′), amino (—NRR′), carboxylicacid (—CO₂H), carboxylic ester (—CO₂R′), carbamoyl (—OC(═O)NR′R″ or—NRC(═O)OR′), hydroxy (—OH), isocyano (—NC), sulfonate (—S(═O)₂OR),sulfonamide (—S(═O)₂NRR′ or —NRS(O)₂R′), or sulfonyl (—S(═O)₂R), whereeach R or R′ is selected, independently, from H, C1-C6 alkyl orheteroaryl, C2-C6 alkenyl or heteroalkenyl, 2C-6C alkynyl orheteroalkynyl, aryl, or heteroaryl. A substituted group may have, forexample, 1, 2, 3, 4, 5, 6, 7, 8, or 9 substituents.

Typical optional substituents on aromatic or heteroaromatic groupsinclude independently halo, CN, NO₂, CF₃. OCF₃, COOR′, CONR′₂, OR′, SR′,SOR′, SO₂R′, NR′₂, NR′(CO)R′, NR′C(O)OR′, NR′C(O)NR′₂, NR′SO₂NR′₂, orNR′SO₂R′, wherein each R′ is independently H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, heteroaryl, and aryl (all as definedabove); or the substituent may be an optionally substituted groupselected from alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,heteroalkynyl, aryl, heteroaryl, O-aryl, O-heteroaryl and arylalkyl.

Optional substituents on a non-aromatic group (e.g., alkyl, alkenyl, andalkynyl groups), are typically selected from the same list ofsubstituents suitable for aromatic or heteroaromatic groups, except asnoted otherwise herein. A non-aromatic group may also include asubstituent selected from ═O and ═NOR′ where R′ is H or an optionallysubstituted group selected from alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteralkynyl, heteroaryl, and aryl (all as definedabove).

The term an “effective amount” of an agent (e.g., a compound accordingto any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1),as used herein, is that amount sufficient to effect beneficial ordesired results, such as clinical results, and, as such, an “effectiveamount” depends upon the context in which it is being applied. Forexample, in the context of administering an agent that is a modulator ofa voltage-gated ion channel (e.g., a sodium channel such as Na_(v)0.7 orNa_(v) 1.8), an effective amount of an agent is, for example, an amountsufficient to achieve a change in sodium channel activity as compared tothe response obtained without administration of the agent.

The term “pharmaceutical composition,” as used herein, represents acomposition containing a compound described herein (e.g., a compoundaccording to any of Formulas (I)-(XIII) or any of Compounds (1)-(236) ofTable 1) formulated with a pharmaceutically acceptable excipient. Insome embodiments, the pharmaceutical composition is manufactured or soldwith the approval of a governmental regulatory agency as part of atherapeutic regimen for the treatment of disease in a mammal.Pharmaceutical compositions can be formulated, for example, for oraladministration in unit dosage form (e.g., a tablet, capsule, caplet,gelcap, or syrup); for topical administration (e.g., as a cream, gel,lotion, or ointment); for intravenous administration (e.g., as a sterilesolution free of particulate emboli and in a solvent system suitable forintravenous use); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, refers anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, or waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc,titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term “pharmaceutically acceptable prodrugs” as used herein,represents those prodrugs of the compounds of the present invention thatare, within the scope of sound medical judgment, suitable for use incontact with the tissues of humans and animals with undue toxicity,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio, and effective for their intended use, aswell as the zwitterionic forms, where possible, of the compounds of theinvention.

The term “pharmaceutically acceptable salt,” as use herein, representsthose salts of the compounds described here (e.g., a compound accordingto any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1)that are, within the scope of sound medical judgment, suitable for usein contact with the tissues of humans and animals without unduetoxicity, irritation, allergic response and the like and arecommensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds described herein orseparately by reacting the free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so as to becapable of preparation as pharmaceutically acceptable salts. These saltsmay be acid addition salts involving inorganic or organic acids or thesalts may, in the case of acidic forms of the compounds of the inventionbe prepared from inorganic or organic bases. Frequently, the compoundsare prepared or used as pharmaceutically acceptable salts prepared asaddition products of pharmaceutically acceptable acids or bases.Suitable pharmaceutically acceptable acids and bases are well-known inthe art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic,citric, or tartaric acids for forming acid addition salts, and potassiumhydroxide, sodium hydroxide, ammonium hydroxide, caffeine, variousamines, and the like for forming basic salts. Methods for preparation ofthe appropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts andthe like. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamineand the like.

The term “pharmaceutically acceptable solvate” as used herein means acompound as described herein (e.g., a compound according to any ofFormulas (I)-(XII) or any of Compounds (1)-(236) of Table 1) wheremolecules of a suitable solvent are incorporated in the crystal lattice.A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the moleculeis referred to as a “hydrate.”

The term “prevent,” as used herein, refers to prophylactic treatment ortreatment that prevents one or more symptoms or conditions of a disease,disorder, or conditions described herein (for example, pain (e.g.,chronic or acute pain), epilepsy, Alzheimer's disease, Parkinson'sdisease, cardiovascular disease, diabetes, cancer, sleep disorders,obesity, psychosis such as schizophrenia, overactive bladder, renaldisease, neuroprotection, addiction, and male birth control).Preventative treatment can be initiated, for example, prior to(“pre-exposure prophylaxis”) or following (“post-exposure prophylaxis”)an event that precedes the onset of the disease, disorder, orconditions. Preventive treatment that includes administration of acompound described herein (e.g., a compound according to any of Formulas(I)-(XIII) or any of Compounds (1)-(236) of Table 1), or apharmaceutically acceptable salt or solvate thereof, or a pharmaceuticalcomposition thereof, can be acute, short-term, or chronic. The dosesadministered may be varied during the course of preventative treatment.

The term “prodrug,” as used herein, represents compounds that arerapidly transformed in vivo to the parent compound of the above formula,for example, by hydrolysis in blood. Prodrugs of the compounds describedherein may be conventional esters. Some common esters that have beenutilized as prodrugs are phenyl esters, aliphatic (C1-C8 or C8-C24)esters, cholesterol esters, acyloxymethyl esters, carbamates, and aminoacid esters. For example, a compound that contains an OH group may beacylated at this position in its prodrug form. A thorough discussion isprovided in T. Higuchi and V. Stella, Pro-drugs as Novel DeliverySystems, Vol. 14 of the A.C.S. Symposium Series, Edward B. Roche, ed.,Bioreversible Carriers in Drug Design, American PharmaceuticalAssociation and Pergamon Press, 1987, and Judkins et al., SyntheticCommunications 26(23):4351-4367, 1996, each of which is incorporatedherein by reference. Preferably, prodrugs of the compounds of thepresent invention are suitable for use in contact with the tissues ofhumans and animals with undue toxicity, irritation, allergic response,and the like, commensurate with a reasonable benefit/risk ratio, andeffective for their intended use.

In addition, the compounds of the invention may be coupled throughconjugation to substances designed to alter the pharmacokinetics, fortargeting, or for other reasons. Thus, the invention further includesconjugates of these compounds. For example, polyethylene glycol is oftencoupled to substances to enhance half-life; the compounds may be coupledto liposomes covalently or noncovalently or to other particulatecarriers. They may also be coupled to targeting agents such asantibodies or peptidomimetics, often through linker moieties. Thus, theinvention is also directed to compounds (e.g., a compound according toany of Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1)when modified so as to be included in a conjugate of this type.

As used herein, and as well understood in the art, “to treat” acondition or “treatment” of the condition (e.g., the conditionsdescribed herein such as pain (e.g., chronic or acute pain), epilepsy,Alzheimer's disease, Parkinson's disease, cardiovascular disease,diabetes, cancer, sleep disorders, obesity, psychosis such asschizophrenia, overactive bladder, renal disease, neuroprotection,addiction, and male birth control) is an approach for obtainingbeneficial or desired results, such as clinical results. Beneficial ordesired results can include, but are not limited to, alleviation oramelioration of one or more symptoms or conditions; diminishment ofextent of disease, disorder, or condition; stabilized (i.e., notworsening) state of disease, disorder, or condition; preventing spreadof disease, disorder, or condition; delay or slowing the progress of thedisease, disorder, or condition; amelioration or palliation of thedisease, disorder, or condition; and remission (whether partial ortotal), whether detectable or undetectable. “Palliating” a disease,disorder, or condition means that the extent and/or undesirable clinicalmanifestations of the disease, disorder, or condition are lessenedand/or time course of the progression is slowed or lengthened, ascompared to the extent or time course in the absence of treatment.

The term “unit dosage form” refers to a physically discrete unitsuitable as a unitary dosage for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with anysuitable pharmaceutical excipient or excipients. Exemplary, non-limitingunit dosage forms include a tablet (e.g., a chewable tablet), caplet,capsule (e.g., a hard capsule or a soft capsule), lozenge, film, strip,gelcap, and syrup.

In some cases, the compounds of the invention contain one or more chiralcenters. The invention includes each of the isolated stereoisomericforms as well as mixtures of stereoisomers in varying degrees of chiralpurity, including racemic mixtures. It also encompasses the variousdiastereomers and tautomers that can be formed.

Compounds useful in the invention may also be isotopically labeledcompounds. Useful isotopes include hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, (e.g., ²H, ³H, ¹³C, ¹⁴C, ¹³N, ¹⁸O,¹⁷O, ³¹P, ³², ³⁵S, ¹⁸F, and ³Cl). Isotopically labeled compounds can beprepared by synthesizing a compound using a readily availableisotopically labeled reagent in place of a non-isotopically labeledreagent. In some embodiments, the compound (e.g., a compound accordingto any of Formulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1),or a composition that includes the compound, has the natural abundanceof each element present in the compound.

The compounds described herein (e.g., a compound according to any ofFormulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1) are alsouseful for the manufacture of a medicament useful to treat conditionsrequiring modulation of voltage-gated ion channel activity (e.g., sodiumchannel activity), and, in particular, Na_(v) 1.7 or Na_(v) 1.8 channelactivity.

Other features and advantages of the invention will be apparent from thefollowing detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show the modulation of ion channel activity by the compoundsdescribed herein.

FIGS. 2A-2C and 3A-3C show data obtained in the spinal nerve ligation(SNL) assay for select compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION Compounds

The invention features compounds that can modulate the activity ofvoltage-gated ion channels (e.g., voltage-gated sodium channels). Thesecompounds can also be used to treat disorders such as pain, epilepsy,Parkinson's disease, mood disorders, psychosis (e.g., schizophrenia),tinnitus, amyotropic lateral sclerosis, glaucoma, ischaemia, spasticitydisorders, obsessive compulsive disorder, restless leg syndrome, andTourette syndrome. Exemplary compounds described herein includecompounds that have a structure according to the following formula,

or a pharmaceutically acceptable salt or solvate thereof, where each ofR¹, R², R³, and R⁴ is selected, independently, from H, optionallysubstituted C1-C6 alkyl, optionally substituted C1-C6 haloalkyl,optionally substituted C6-C10 aryl, or optionally substituted 5 to6-membered heteroaryl, where at least one of R¹, R², R³, and R ishalogen or optionally substituted C1-C6 haloalkyl;

R⁵ is H, optionally substituted C1-C6 alkyl, or optionally substitutedC1-C10 heteroalkyl;

R⁶ is —R^(6A) or —CH₂R^(6B);

R^(6A) is NH₂, optionally substituted cyclopropyl, optionallysubstituted azetidine, optionally substituted cyclopentyl, optionallysubstituted pyrazole, optionally substituted pyrrole, optionallysubstituted pyrrolidine, optionally substituted thiazolidine, optionallysubstituted thiazolidine-1,1-dioxide, optionally substituted pyrimidine,optionally substituted C1-C10 aminoalkyl, optionally substituted C1-C10hydroxyalkyl, optionally substituted C1-C10 alkoxyalkyl, optionallysubstituted C1-C10 haloalkyl, or optionally substituted C1-C10alkylsulfonyl; or R^(6A) has a structure according to

where

n is an integer between 0-4;

Z¹ is CH₂, NH, NCH₃, or O;

L¹ is —CH₂, —CHR^(A), —CH₂C(═O), —C(═O)CH₂, —CH₂C(═O)NH, —CH₂C(═O)NHCH₂,or —CH₂NHC(═O)CH₂;

-   -   each R^(2A) and R^(2C), when present, is selected from OH,        N(R^(2B))₂, halogen, and unsubstituted C1-C3 alkyl, or two        R^(2A) combine to form an oxo (═O) group, and wherein no more        than two R^(2A) combine to form an oxo group; and

each R^(2B) is, independently, H or unsubstituted C1-C6 alkyl;

R^(2D) is H, OH, or NH₂;

and

R^(6B) is optionally substituted cyclopropyl, optionally substitutedazetidine, optionally substituted cyclopentyl, optionally substitutedpyrazole, optionally substituted pyrrole, optionally substitutedpyrrolidine, optionally substituted thiazolidine, optionally substitutedthiazolidine-1,1-dioxide, or optionally substituted pyrimidine.

In some embodiments, R⁵ is H.

In other embodiments, R⁵ is optionally substituted C1-C10 heteroalkyl.

In certain embodiments, R² and R⁴ are both CF₃, F, or Cl.

In still other embodiments, R² and R³ are both CF₃, F, or Cl.

In some embodiments, R⁶ is —CH₂R^(6B), and R^(6B) is optionallysubstituted azetidine.

In certain embodiments, R⁶ is optionally substituted C1-C10 aminoalkyl.

In other embodiments, the C1-C10 aminoalkyl includes an oxo (═O)substituent, an alkoxy substituent, an N-sulfonyl group, or anycombination thereof.

Other embodiments (e.g., Formulas (II)-(XIII) and any of compounds(1)-(236) of Table 1), as well as exemplary methods for the synthesis ofthese compounds, are described herein.

Utility and Administration

The compounds described herein (e.g., a compound according to any ofFormulas (I)-(XII) or any of Compounds (1)-(236) of Table 1) are usefulin the methods of the invention and, while not bound by theory, arebelieved to exert their desirable effects through their ability tomodulate the activity of voltage-gated ion channels, e.g., the activityof sodium channels such as the Na_(v) 1.7 and Na_(v)1.8 channels. Thecompounds described herein (e.g., a compound according to any ofFormulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1) can alsobe used for the treatment of certain conditions such as pain, epilepsy,migraine, Parkinson's disease, mood disorders, schizophrenia, psychosis,tinnitus, amyotropic lateral sclerosis, glaucoma ischaemia, spasticitydisorders, obsessive compulsive disorder, restless leg syndrome, andTourette syndrome.

Modulation of Sodium Channels

There are nine Na_(v)1 α-subunit isoforms: Na_(v)1.1-1.9 (see, e.g., Yuet al., Genome Biolog, 4:207, 2003). In addition to pain, otherconditions associated with voltage-dependent sodium channel activityinclude seizures (e.g., Na_(v)1.1), epilepsy (e.g., Na_(v)1.2),neurodegeneration (e.g., Na_(v)1.1, Na_(v)1.2), myotonia (e.g.,Na_(v)1.4), arrhythmia (e.g., Na_(v)1.5), and movement disorders (e.g.,Na_(v)1.6) as described in PCT Publication No. WO 2008/118758, hereinincorporated by reference. The expression of particular isoforms inparticular tissues can influence the therapeutic effects of sodiumchannel modulators. For example, the Na_(v)1.4 and Na_(v)1.5 isoformsare largely found in skeletal and cardiac myocytes (see, e.g., Gold, ExpNeurol. 210(1): 1-6, 2008).

Sodium Channel Activity and Pain

Voltage-dependent ion channels in pain-sensing neurons are currently ofgreat interest in developing drugs to treat pain. For example, blockingvoltage-dependent sodium channels in pain-sensing neurons can block painsignals by interrupting initiation and transmission of the actionpotential. Studies also indicate that particular sodium channel isoformsare predominantly expressed in peripheral sensory neurons associatedwith pain sensation; for example, Na_(v)1.7, Na_(v)1.8 and Na_(v) 1.9activity are thought to be involved in inflammatory, and possiblyneuropathic, pain (see, e.g., Cummins et al., Pain, 131(3):243-257,2007). The Na_(v)1.3 isoform has also been implicated in pain, e.g.,pain associated with tissue injury (Gold, Exp Neurol. 210(1): 1-6,2008).

The Na_(v)1.7 and Na_(v)1.8 channel subtypes act as major contributorsto both inflammatory and neuropathic pain (vide infra). Recently,mutations have been identified in the Na_(v)1.7 channel that lead eitherto a gain of channel function (Dib-Hajj et al., Brain 128:1847-1854,2005) or more commonly to a loss of channel function (Chatelier et al.,J. Neurophisiol. 99:2241-50, 2008). These mutations underlie humanheritable disorders such as erythermalgia (Yang et al., J Med Genet.41(3) 171-4, 2004), paroxysmal extreme pain disorder (Fertleman et al.,Neuron. 52(5) 767-74, 2006), and congenital indifference to pain (Cox etal., Nature 444(7121):894-8, 2006). Behavioral studies have shown inmice that inflammatory and acute mechanosensory pain is reduced whenNa_(v)1.7 is knocked out in Na_(v)1.8-positive neurons (Nassar et al.,Proc Natl Acad Sci USA. 101(34):12706-11, 2004). In addition, siRNA ofNa_(v)1.7 attenuates inflammatory hyperalgesia (Yeomans et al., Hum GeneTher. 16(2) 271-7, 2005).

The Na_(v)1.8 isoform is selectively expressed in sensory neurons andhas been identified as a target for the treatment of pain, e.g., chronicpain (e.g., Swanwick et al., Neurosci. Lett. 486:78-83, 2010). The roleof Na_(v)1.8 in inflammatory (Khasar et al., Neurosci Lett.256(1):17-20, 1998), neuropathic and mechanical hyperalgesia (Joshi etal., Pain 123(1-2):75-82, 2006) has also emerged using moleculartechniques to knockdown Na_(v) 1.8, which has been shown to reduce themaintenance of these different pain states.

Lacosamide is a functionalized amino acid that has shown effectivenessas an analgesic in several animal models of neuropathic pain and iscurrently in late stages of clinical development for epilepsy anddiabetic neuropathic pain. One mode of action that has been validatedfor lacosamide is inhibition of voltage-gated sodium channel activity byselective inhibition with the slow-inactivated conformation of thechannel (Sheets et al., Journal of Pharmacology and ExperimentalTherapeutics, 326(1) 89-99 (2008)). Modulators of sodium channels,including clinically relevant compounds, can exhibit a pronouncedstate-dependent binding, where sodium channels that are rapidly andrepeatedly activated and inactivated are more readily blocked. In asimplified scheme, voltage-gated sodium channels have four distinctstates: open, closed, fast-inactivated and slow-inactivated. Classicsodium channel modulators, such as lidocaine, are believed to exhibitthe highest affinity for the fast-inactivated state. However, alterationof the slow inactivated state is also clinically relevant.

Modulation of Calcium Channels

The entry of calcium into cells through voltage-gated calcium channelsmediates a wide variety of cellular and physiological responses,including excitation-contraction coupling, hormone secretion and geneexpression (e.g., Miller et al., Science 235:46-52 (1987); Augustine etal., Annu Rev Neurosci 10: 633-693 (1987)). In neurons, calcium channelsdirectly affect membrane potential and contribute to electricalproperties such as excitability, repetitive firing patterns andpacemaker activity. Calcium entry further affects neuronal functions bydirectly regulating calcium-dependent ion channels and modulating theactivity of calcium-dependent enzymes such as protein kinase C andcalmodulin-dependent protein kinase II. An increase in calciumconcentration at the presynaptic nerve terminal triggers the release ofneurotransmitter, which also affects neurite outgrowth and growth conemigration in developing neurons.

Calcium channels mediate a variety of normal physiological functions,and are also implicated in a number of human disorders as describedherein. For example, calcium channels also have been shown to mediatethe development and maintenance of the neuronal sensitization andhyperexcitability processes associated with neuropathic pain, andprovide attractive targets for the development of analgesic drugs(reviewed in Vanegas et al., Pain 85: 9-18 (2000)). Native calciumchannels have been classified by their electrophysiological andpharmacological properties into T-, L-, N-, P/Q- and R-types (reviewedin Catterall, Annu Rev Cell Dev Biol 16: 521-555, 2000; Huguenard, AnnuRev Physiol 58: 329-348, 1996). The L-, N- and P/Q-type channelsactivate at more positive potentials (high voltage-activated) anddisplay diverse kinetics and voltage-dependent properties (Id.). T-typechannels can be distinguished by having a more negative range ofactivation and inactivation, rapid inactivation, slow deactivation, andsmaller single-channel conductances. There are three subtypes of T-typecalcium channels that have been molecularly, pharmacologically, andelecrophysiologically identified: these subtypes have been termedα_(1G), α_(1H), and α_(1I) (alternately called Cav 3.1, Cav 3.2 and Cav3.3 respectively).

T-type calcium channels are involved in various medical conditions. Inmice lacking the gene expressing the 3.1 subunit, resistance to absenceseizures was observed (Kim et al., Mol. Cell Neurosci. 18(2): 235-245(2001)). Other studies have also implicated the 3.2 subunit in thedevelopment of epilepsy (Su et al., J. Neurosci. 22: 3645-3655 (2002)).There is also evidence that some existing anticonvulsant drugs, such asethosuximide, function through the blockade of T-type channels (Gomoraet al., Mol. Pharmacol. 60: 1121-1132 (2001)).

Low voltage-activated calcium channels are highly expressed in tissuesof the cardiovascular system. There is also a growing body of evidencethat suggests that T-type calcium channels are abnormally expressed incancerous cells and that blockade of these channels may reduce cellproliferation in addition to inducing apoptosis. Recent studies alsoshow that the expression of T-type calcium channels in breast cancercells is proliferation state dependent, i.e. the channels are expressedat higher levels during the fast-replication period, and once the cellsare in a non-proliferation state, expression of this channel is minimal.Therefore, selectively blocking calcium channel entry into cancerouscells may be a valuable approach for preventing tumor growth (e.g., PCTPatent Application Nos. WO 05/086971 and WO 05/77082; Taylor et al.,World J. Gastroenterol. 14(32): 4984-4991 (2008); Heo et al., Biorganic& Medicinal Chemistry Letters 18:3899-3901 (2008)).

T-type calcium channels may also be involved in still other conditions.A recent study also has shown that T-type calcium channel antagonistsinhibit high-fat diet-induced weight gain in mice. In addition,administration of a selective T-type channel antagonist reduced bodyweight and fat mass while concurrently increasing lean muscle mass(e.g., Uebele et al., The Journal of Clinical Investigation,119(6):1659-1667 (2009)). T-type calcium channels may also be involvedin pain (see for example: US Patent Publication No. 2003/0086980; PCTPublication Nos. WO 03/007953 and WO 04/000311). In addition tocardiovascular disease, epilepsy (see also US Patent Application No.2006/0025397), cancer, and chronic or acute pain, T-type calciumchannels have been implicated in diabetes (US Patent Publication No.2003/0125269), sleep disorders (US Patent Publication No. 2006/0003985),Parkinson's disease and psychosis such as schizophrenia (US PatentPublication No. 2003/0087799); overactive bladder (Sui et al., BritishJournal of Urology International 99(2): 436-441 (2007); US PatentPublication No. 2004/0197825), renal disease (Hayashi et al., Journal ofPharmacological Sciences 99: 221-227 (2005)), anxiety and alcoholism (USPatent Publication No. 2009/0126031), neuroprotection, and male birthcontrol.

The modulation of ion channels by the compounds described herein (e.g.,a compound according to any of Formulas (I)-(XIII) or any of Compounds(1)-(236) of Table 1) can be measured according to methods known in theart (e.g., in the references provided herein). Modulators of ionchannels, e.g., voltage gated sodium and calcium ion channels, and themedicinal chemistry or methods by which such compounds can beidentified, are also described in, for example: Birch et al., DrugDiscovery Today, 9(9):410-418 (2004); Audesirk, “Chapter6-Electrophysiological Analysis of Ion Channel Function,”Neurotoxicology: Approaches and Methods, 137-156 (1995); Camerino etal., “Chapter 4: Therapeutic Approaches to Ion Channel Diseases,”Advances in Genetics, 64:81-145 (2008); Petkov, “Chapter 16—IonChannels,” Pharmacology: Principles and Practice, 387427 (2009); Standenet al., “Chapter 15—Patch Clamping Methods and Analysis of IonChannels,” Principles of Medical Biology, Vol. 7, Part 2, 355-375(1997); Xu et al., Drug Discovery Today, 6(24):1278-1287 (2001); andSullivan et al., Methods Mol. Biol. 114:125-133 (1999). Exemplaryexperimental methods are also provided in the Examples.

Diseases and Conditions

Exemplary conditions that can be treated using the compounds describedherein include pain (e.g., chronic or acute pain), epilepsy. Alzheimer'sdisease. Parkinson's disease, diabetes; cancer; sleep disorders;obesity; mood disorders, psychosis such as schizophrenia; overactivebladder, renal disease, neuroprotection, and addiction. For example, thecondition can be pain (e.g., neuropathic pain or post-surgery pain),epilepsy, migraine, Parkinson's disease, mood disorders, schizophrenia,psychosis, tinnitus, amyotropic lateral sclerosis, glaucoma, ischaemia,spasticity disorders, obsessive compulsive disorder, restless legsyndrome, and Tourette syndrome.

Epilepsy as used herein includes but is not limited to partial seizuressuch as temporal lobe epilepsy, absence seizures, generalized seizures,and tonic/clonic seizures.

Cancer as used herein includes but is not limited to breast carcinoma,neuroblastoma, retinoblastoma, glioma, prostate carcinoma, esophagealcarcinoma, fibrosarcoma, colorectal carcinoma, pheochromocytoma,adrenocarcinoma, insulinoma, lung carcinoma, melanoma, and ovariancancer.

Acute pain as used herein includes but is not limited to nociceptivepain and post-operative pain. Chronic pain includes but is not limitedby: peripheral neuropathic pain (e.g., post-herpetic neuralgia, diabeticneuropathic pain, neuropathic cancer pain, HIV-associated neuropathy,erythromelalgia, failed back-surgery syndrome, trigeminal neuralgia, orphantom limb pain); central neuropathic pain (e.g., multiple sclerosisrelated pain, Parkinson disease related pain, post-stroke pain,post-traumatic spinal cord injury pain, lumbosacral radiculopathy,cervical radiculopathy, brachial radiculopathy, or pain in dementia);musculoskeletal pain such as osteoarthritic pain and fibromyalgiasyndrome; inflammatory pain (e.g., inflammatory pain caused byrheumatoid arthritis, juvenile idiopathic arthritis, ankylosingspondylitis, psoriatic arthritis, inflammatory bowel disease, primarydysmenorrhea, or endometriosis); headache such as migraine, clusterheadache, tension headache syndrome, facial pain, headache caused byother diseases; visceral pain such as interstitial cystitis, irritablebowel syndrome and chronic pelvic pain syndrome; and mixed pain such aslower back pain, neck and shoulder pain, burning mouth syndrome andcomplex regional pain syndrome.

In treating osteoarthritic pain, joint mobility can also improve as theunderlying chronic pain is reduced. Thus, use of compounds of thepresent invention to treat osteoarthritic pain inherently includes useof such compounds to improve joint mobility in patients suffering fromosteoarthritis.

The compounds described herein can be tested for efficacy in anystandard animal model of pain. Various models test the sensitivity ofnormal animals to intense or noxious stimuli (physiological ornociceptive pain). These tests include responses to thermal, mechanical,or chemical stimuli. Thermal stimuli usually involve the application ofhot stimuli (typically varying between 42-55° C.) including, forexample: radiant heat to the tail (the tail flick test), radiant heat tothe plantar surface of the hindpaw (the Hargreaves test), the hotplatetest, and immersion of the hindpaw or tail into hot water. Immersion incold water, acetone evaporation, or cold plate tests may also be used totest cold pain responsiveness. Tests involving mechanical stimulitypically measure the threshold for eliciting a withdrawal reflex of thehindpaw to graded strength monofilament von Frey hairs or to a sustainedpressure stimulus to a paw (e.g., the Ugo Basile analgesiometer). Theduration of a response to a standard pinprick may also be measured. Whenusing a chemical stimulus, the response to the application or injectionof a chemical irritant (e.g., capsaicin, mustard oil, bradykinin, ATP,formalin, acetic acid) to the skin, muscle joints or internal organs(e.g., bladder or peritoneum) is measured.

In addition, various tests assess pain sensitization by measuringchanges in the excitability of the peripheral or central components ofthe pain neural pathway. In this regard, peripheral sensitization (i.e.,changes in the threshold and responsiveness of high thresholdnociceptors) can be induced by repeated heat stimuli as well as theapplication or injection of sensitizing chemicals (e.g., prostaglandins,bradykinin, histamine, serotonin, capsaicin, or mustard oil). Centralsensitization (i.e., changes in the excitability of neurons in thecentral nervous system induced by activity in peripheral pain fibers)can be induced by noxious stimuli (e.g., heat), chemical stimuli (e.g.,injection or application of chemical irritants), or electricalactivation of sensory fibers.

Various pain tests developed to measure the effect of peripheralinflammation on pain sensitivity can also be used to study the efficacyof the compounds (Stein et al., Pharmacol. Biochem. Behav. (1988) 31:445-451; Woolf et al., Neurosci. (1994) 62: 327-331). Additionally,various tests assess peripheral neuropathic pain using lesions of theperipheral nervous system. One such example is the “axotomy pain model”(Watson, J. Physiol. (1973) 231:41). Other similar tests include the SNLtest which involves the ligation of a spinal segmental nerve (Kim andChung Pain (1992) 50: 355), the Seltzer model involving partial nerveinjury (Seltzer, Pain (1990)43: 205-18), the spared nerve injury (SNI)model (Decosterd and Woolf, Pain (2000) 87:149), chronic constrictioninjury (CCI) model (Bennett (1993) Muscle Nerve 16: 1040), testsinvolving toxic neuropathies such as diabetes (streptozocin model),pyridoxine neuropathy, taxol, vincristine, and other antineoplasticagent-induced neuropathies, tests involving ischaemia to a nerve,peripheral neuritis models (e.g., CFA applied peri-neurally), models ofpost-herpetic neuralgia using HSV infection, and compression models.

In all of the above tests, outcome measures may be assessed, forexample, according to behavior, electrophysiology, neurochemistry, orimaging techniques to detect changes in neural activity.

Exemplary models of pain are also described in the Examples providedherein.

In addition to being able to modulate a particular voltage-gated ionchannel, e.g., a sodium channel, it may be desirable that the compoundhas very low activity with respect to the hERG K⁺ channel, which isexpressed in the heart: compounds that block this channel with highpotency may cause reactions which are fatal. See, e.g., Bowlby et al.,“hERG (KCNH2 or K_(v)11.1 K⁺ Channels: Screening for Cardiac ArrhythmiaRisk,” Curr. Drug Metab. 9(9):965-70 (2008)). Thus, for a compound thatmodulates sodium channel activity, it may also be shown that the hERG K⁺channel is not inhibited or only minimally inhibited as compared to theinhibition of the primary channel targeted. Similarly, it may bedesirable that the compound does not inhibit cytochrome p450, an enzymethat is required for drug detoxification. Such compounds may beparticularly useful in the methods described herein.

Pharmaceutical Compositions

For use as treatment of human and animal subjects, the compounds of theinvention can be formulated as pharmaceutical or veterinarycompositions. Depending on the subject to be treated, the mode ofadministration, and the type of treatment desired—e.g., prevention,prophylaxis, or therapy—the compounds are formulated in ways consonantwith these parameters. A summary of such techniques is found inRemington: The Science and Practice of Pharmacy, 21^(st) Edition,Lippincott Williams & Wilkins, (2005); and Encyclopedia ofPharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,1988-1999, Marcel Dekker, New York, each of which is incorporated hereinby reference.

The compounds described herein (e.g., a compound according to any ofFormulas (I)-(XIII) or any of Compounds (1)-(236) of Table 1) may bepresent in amounts totaling 1-95% by weight of the total weight of thecomposition. The composition may be provided in a dosage form that issuitable for intraarticular, oral, parenteral (e.g., intravenous,intramuscular), rectal, cutaneous, subcutaneous, topical, transdermal,sublingual, nasal, vaginal, intravesicular, intraurethral, intrathecal,epidural, aural, or ocular administration, or by injection, inhalation,or direct contact with the nasal, genitourinary, gastrointestinal,reproductive or oral mucosa. Thus, the pharmaceutical composition may bein the form of, e.g., tablets, capsules, pills, powders, granulates,suspensions, emulsions, solutions, gels including hydrogels, pastes,ointments, creams, plasters, drenches, osmotic delivery devices,suppositories, enemas, injectables, implants, sprays, preparationssuitable for iontophoretic delivery, or aerosols. The compositions maybe formulated according to conventional pharmaceutical practice.

In general, for use in treatment, the compounds described herein (e.g.,a compound according to any of Formulas (I)-(XII) or any of Compounds(1)-(236) of Table 1) may be used alone, as mixtures of two or morecompounds or in combination with other pharmaceuticals. An example ofother pharmaceuticals to combine with the compounds described herein(e.g., a compound according to any of Formulas (I)-(XII) or any ofCompounds (1)-(236) of Table 1) would include pharmaceuticals for thetreatment of the same indication. For example, in the treatment of pain,a compound may be combined with another pain relief treatment such as anNSAID, or a compound which selectively inhibits COX-2, or an opioid, oran adjuvant analgesic such as an antidepressant. Another example of apotential pharmaceutical to combine with the compounds described herein(e.g., a compound according to any of Formulas (I)-(XIII) or any ofCompounds (1)-(236) of Table 1) would include pharmaceuticals for thetreatment of different yet associated or related symptoms orindications. Depending on the mode of administration, the compounds willbe formulated into suitable compositions to permit facile delivery. Eachcompound of a combination therapy may be formulated in a variety of waysthat are known in the art. For example, the first and second agents ofthe combination therapy may be formulated together or separately.Desirably, the first and second agents are formulated together for thesimultaneous or near simultaneous administration of the agents.

The compounds of the invention may be prepared and used aspharmaceutical compositions comprising an effective amount of a compounddescribed herein (e.g., a compound according to any of Formulas(I)-(XIII) or any of Compounds (1)-(236) of Table 1) and apharmaceutically acceptable carrier or excipient, as is well known inthe art. In some embodiments, the composition includes at least twodifferent pharmaceutically acceptable excipients or carriers.

Formulations may be prepared in a manner suitable for systemicadministration or topical or local administration. Systemic formulationsinclude those designed for injection (e.g., intramuscular, intravenousor subcutaneous injection) or may be prepared for transdermal,transmucosal, or oral administration. The formulation will generallyinclude a diluent as well as, in some cases, adjuvants, buffers,preservatives and the like. The compounds can be administered also inliposomal compositions or as microemulsions.

For injection, formulations can be prepared in conventional forms asliquid solutions or suspensions or as solid forms suitable for solutionor suspension in liquid prior to injection or as emulsions. Suitableexcipients include, for example, water, saline, dextrose, glycerol andthe like. Such compositions may also contain amounts of nontoxicauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and the like, such as, for example, sodium acetate, sorbitanmonolaurate, and so forth.

Various sustained release systems for drugs have also been devised. See,for example, U.S. Pat. No. 5,624,677, which is herein incorporated byreference.

Systemic administration may also include relatively noninvasive methodssuch as the use of suppositories, transdermal patches, transmucosaldelivery and intranasal administration. Oral administration is alsosuitable for compounds of the invention. Suitable forms include syrups,capsules, and tablets, as is understood in the art.

For administration to animal or human subjects, the dosage of thecompounds of the invention may be, for example, 0.01-50 mg/kg (e.g.,0.01-15 mg/kg or 0.1-10 mg/kg). For example, the dosage can be 10-30mg/kg.

Each compound of a combination therapy, as described herein, may beformulated in a variety of ways that are known in the art. For example,the first and second agents of the combination therapy may be formulatedtogether or separately.

The individually or separately formulated agents can be packagedtogether as a kit. Non-limiting examples include, but are not limitedto, kits that contain, e.g., two pills, a pill and a powder, asuppository and a liquid in a vial, two topical creams, etc. The kit caninclude optional components that aid in the administration of the unitdose to patients, such as vials for reconstituting powder forms,syringes for injection, customized IV delivery systems, inhalers, etc.Additionally, the unit dose kit can contain instructions for preparationand administration of the compositions. The kit may be manufactured as asingle use unit dose for one patient, multiple uses for a particularpatient (at a constant dose or in which the individual compounds mayvary in potency as therapy progresses); or the kit may contain multipledoses suitable for administration to multiple patients (“bulkpackaging”). The kit components may be assembled in cartons, blisterpacks, bottles, tubes, and the like.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystallinecellulose, starches including potato starch, calcium carbonate, sodiumchloride, lactose, calcium phosphate, calcium sulfate, or sodiumphosphate); granulating and disintegrating agents (e.g., cellulosederivatives including microcrystalline cellulose, starches includingpotato starch, croscarmellose sodium, alginates, or alginic acid);binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid,sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

Two or more compounds may be mixed together in a tablet, capsule, orother vehicle, or may be partitioned. In one example, the first compoundis contained on the inside of the tablet, and the second compound is onthe outside, such that a substantial portion of the second compound isreleased prior to the release of the first compound.

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent (e.g., potato starch, lactose, microcrystallinecellulose, calcium carbonate, calcium phosphate or kaolin), or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example, peanut oil, liquid paraffin, or olive oil.Powders, granulates, and pellets may be prepared using the ingredientsmentioned above under tablets and capsules in a conventional mannerusing, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Dissolution or diffusion controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Generally, when administered to a human, the oral dosage of any of thecompounds of the combination of the invention will depend on the natureof the compound, and can readily be determined by one skilled in theart. Typically, such dosage is normally about 0.001 mg to 2000 mg perday, desirably about 1 mg to 1000 mg per day, and more desirably about 5mg to 500 mg per day. Dosages up to 200 mg per day may be necessary.

Administration of each drug in a combination therapy, as describedherein, can, independently, be one to four times daily for one day toone year, and may even be for the life of the patient. Chronic,long-term administration may be indicated.

Synthesis

The reaction scheme and Examples are intended to illustrate thesynthesis of a representative number of compounds. Accordingly, theExamples are intended to illustrate but not to limit the invention.Additional compounds not specifically exemplified may be synthesizedusing conventional methods in combination with the methods describedherein.

EXAMPLES Example 1. Synthesis of(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl) methanamine (5)

Synthesis of tert-butyl(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)carbamate(3)

3,5-Bis(trifluoromethyl)benzene-1,2-diamine (1) (3.0 g, 12.3 mol),2-((tert-butoxycarbonyl)amino)acetic acid (2) (2.1 g, 12.3 mmol), HATU(6.4 g, 17.2 mmol), and triethylamine (TEA; 3.5 mL, 25 mmol) werestirred in dichloromethane (DCM; 50 mL) at room temperature for 17hours. The reaction was diluted with DCM (100 mL), washed sequentiallywith NH₄Cl (saturated solution), NaHCO₃ (saturated solution), and brine.The organics were separated, dried (Na₂SO₄), and concentrated in vacuo.The residue was purified by automated column chromatography(EtOAc/petroleum ether, 35/65) to give tert-butyl(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)carbamate(3) (3.07 g, 61.7%); Confirmed by LCMS (Positive ion mode).

Synthesis of tert-Butyl((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)carbamate (4)

tert-Butyl(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)carbamate(3) (1.4 g, 3.5 mmol) was heated in THF/AcOH (95/5, 20 mL) using amicrowave at 140° C. for 2.5 hours. The reaction was concentrated invacuo, taken up in EtOAc, and washed with NaHCO₃ (saturated solution) toneutralize. The organic layer was separated, dried (Na₂SO₄), andconcentrated in vacuo. The residue was purified by automated columnchromatography (EtOAc/PE, 50:50) to give tert-Butyl((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)carbamate(4), (730 mg, 54%); confirmed by LCMS (Positive ion mode)).

Synthesis of(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanaminehydrochloride (5)

tert-Butyl((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)carbamate (4)(730 mg, 1.9 mmol) was taken up in EtOAc, and the solution was flushedwith HCl (g) for 5 minutes. The resultant suspension was stirred at roomtemperature for 25 minutes then concentrated in vacuo. The resultantsolid was dried under high vacuum for 14 hours to give(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanaminehydrochloride (5), in a quantitative manner, ¹H NMR (300 mHz, CD₃OD) δ4.57 (s, 2H), 7.81 (s, 1H), 8.21 (s, 1H).

Example 2. Synthesis of2-amino-N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)acetamidehydrochloride (7)

Synthesis of tert-butyl (2-(((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-oxoethyl)carbamate(6)

(4,6-Bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanaminehydrochloride (5) (1 g, 2.6 mmol), BOC-gly (2) (4.55 mg, 2.6 mmol), HATU(1.35 g, 3.64 mmol), and TEA (0.73 mL, 5.2 mmol) were stirred in DCM atroom temperature for 14 hours. The reaction was diluted with DCM (100mL) and washed sequentially with NH₄Cl (saturated solution), NaHCO₃(saturated solution), and brine. The organics were separated, dried(Na₂SO₄), and concentrated in vacuo. The residue was purified byautomated column chromatography (EtOAc/PE, 50/50) to give tert-butyl(2-(((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-oxoethyl)carbamate(6) (732 mg, 64%; Confirmed by LCMS (positive ion mode)).

Synthesis of2-amino-N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)acetamidehydrochloride (7)

tert-Butyl(2-(((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)amino)-2-oxoethyl)carbamate(6) (732 mg, 1.66 mmol) was taken up in EtOAc, and the solution flushedwith HCl (g) for 5 minutes. The suspension was stirred at roomtemperature for 20 minutes, concentrated in vacuo, and the residuepurified by reverse phase HPLC to give2-amino-N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)acetamidehydrochloride (7); ¹H NMR (300 mHz, CD₃OD) δ 3.45 (s, 2H), 5.04 (s, 2H),8.18 (s, 1H), (8.49, S, 1H).

Example 3. Synthesis of1-((4,6-bis(trfluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-(tert-butyl)urea(9)

Synthesis of1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-(tert-butyl)urea(9)

(4,6-Bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanaminehydrochloride (5) (250 mg, 0.88 mmol) and TEA (0.25 mL, 1.8 mmol) werestirred in DCM. tert-Butyl isocyanate (8) (105 μL, 0.9 mmol) was addedand the reaction stirred at room temperature for 1 h. The reaction wasconcentrated in vacuo and the residue purified by reverse phase HPLC togive1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-(tert-butyl)urea(9). Confirmed by LCMS (positive ion mode).

Example 4. Synthesis of5-(3-chloro-4-fluorophenyl)-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole(15)

Preparation of N-(2-amino-5-bromophenyl)-2-(pyrimidin-5-yl)acetamide(12)

To a solution of 4-bromobenzene-1,2-diamine (10) (0.281 g, 1.5 mmol),2-(pyrimidin-5-yl)acetic acid (11) (0.207 g, 1.5 mmol), and HATU (0.741g, 1.95 mmol) in DCM (50 ml) was added triethylamine (0.63 ml, 4.5mmol). The reaction mixture was stirred at room temperature overnight.The solution was washed with saturated sodium bicarbonate (50 ml) andbrine (50 ml). The DCM solution was dried over sodium sulfate andconcentrated. The residue was purified by automated columnchromatography columned using DCM and methanol as eluents. Yield 0.4 g,87%. MS: m/z 306.9 (M+H⁺).

Preparation of 5-bromo-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole(13)

A solution of N-(2-amino-5-bromophenyl)-2-(pyrimidin-5-yl)acetamide (12)(0.4 g, 1.3 mmol) in THF (12 ml) and acetic acid (7 ml) was reacted inthe microwave at 145° C. for 3 hours. The solvents were removed. Theresidue was dissolved in ethyl acetate (50 ml) and washed with saturatedsodium bicarbonate (30 ml) and brine (30 ml). The ethyl acetate solutionwas dried over sodium sulfate and concentrated. The residue was purifiedby automated column chromatography using DCM and methanol as eluents.Yield 0.345 g, 91%. MS: m/z 288.9 (M+H).

Preparation of5-(3-chloro-4-fluorophenyl)-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole(15)

Pd(dppf)₂Cl₂.DCM (0.274 g, 0.336 mmol) was added to a suspension of5-bromo-2-(pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole (13) (0.345 g,1.12 mmol), 3-chloro-4-fluorobenzene boronic acid (14) (0.234 g, 1.34mmol), and sodium carbonate (0.594 g, 5.6 mmol) in ethanol (7 ml) andtoluene (7 ml). The reaction mixture was stirred at 130° C. overnight.The deep brown solution was filtered through Celite and concentrated.The residue was purified by automated column chromatography using DCMand methanol as eluents. Yield 0.15 g, 40%. MS: m/z 338.9 (M+H⁺).

Example 5. Synthesis of(5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanol (21)

Preparation of ethyl 2-((tert-butyldimethylsilyl)oxy)acetate (17)

A solution of ethyl 2-hydroxyacetate (16) (3.12 g, 30 mmol) andimidazole (2.45 g, 36 mmol) in DCM (100 ml) was addedtert-butylchlorodimethylsilane (5.43 g, 36 mmol) at 0° C. and thereaction mixture was stirred at 0° C. for 1 hour. The reaction mixturewas washed with saturated sodium bicarbonate (50 ml) and brine (50 ml).The DCM solution was dried over sodium sulfate and concentrated. Theresidual (17) (6.68 g, 100% yield) was used in the next step withoutfurther purification.

Preparation of 2-((tert-butyldimethylsilyl)oxy)acetic acid (18)

A solution of ethyl 2-((tert-butyldimethylsilyl)oxy)acetate (17) (6.68g, 30 mmol) in methanol (30 ml) was added 2N NaOH solution (30 ml). Themixture was then stirred at room temperature for 2 hours. The reactionmixture was concentrated, diluted with water, and acidified with 2N HClto pH 4-5. The aqueous solution was extracted with ethyl acetate 3times. The combined ethyl acetate solution was washed with brine anddried over sodium sulfate and concentrated. The residual (18) was usedin the next step without further purification. Yield 2.19 g, 38%. MS,m/z 189.1 (M−H⁺).

Preparation ofN-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-((isopropyldimethylsilyl)oxy)acetamide(19)

A solution of 3,5-bis(trifluoromethyl)benzene-1,2-diamine (1) (1.28 g,5.26 mmol), 2-((tert-butyldimethylsilyl)oxy)acetic acid (18) (18, 5.26mmol) and HATU (2.6 g, 6.84 mmol) in DMF (15 ml) was reacted in themicrowave at 80° C. for 2 hours. The reaction mixture was diluted withethyl acetate. The solution was washed with saturated sodium bicarbonateand brine. The ethyl acetate solution was dried over sodium sulfate andconcentrated. The residue (19) was purified by automated columnchromatography using petroleum ether and ethyl acetate as eluents. Yield0.84 g, 40%. MS, m/z 403.0 (M+H⁺).

Preparation of2-(((ter-butyldimethylsilyl)oxy)methyl)-5,7-bis(trifluoromethyl)-1H-benzo[d]imidazole(20)

A solution of N-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-((isopropyldimethylsilyl)oxy)acetamide (19) (0.84 g, 2.09 mmol) in THF (14 ml) andacetic acid (7 ml) was reacted in the microwave at 145° C. for 3 hours.The solvents were removed. The residue was dissolved in ethyl acetate(50 ml) and washed with saturated sodium bicarbonate (30 ml) and brine(30 ml). The ethyl acetate solution was dried over sodium sulfate andconcentrated. The residue (20) was purified by automated columnchromatography using pet ether and ethyl acetate as eluents. Yield 0.5g, 60%. MS, m/z 399.0 (M+H⁺).

Preparation of(5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanol (21)

To a solution of2-(((tert-butyldimethylsilyl)oxy)methyl)-5,7-bis(trifluoromethyl)-1H-benzo[d]imidazole(20) (0.5 g, 1.26 mmol) in THF (15 ml) was added IM TBAF solution in THF(1.7 ml, 1.7 mmol) at 0° C. The reaction mixture was stirred at roomtemperature overnight. The solvent was removed. The residue wasdissolved in ethyl acetate (50 ml) and washed with brine (30 ml). Theethyl acetate solution was dried over sodium sulfate and concentrated.The residue of (34) was purified by automated column chromatographyusing petroleum ether and ethyl acetate as eluents. Yield 0.22 g, 61%.MS, m/z 284.9 (M+H⁺).

Example 6. Synthesis of(1-((4,6-bis(trifluoremethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-methylurea(24)

Preparation of(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-methylurea(24)

A solution of1-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-N-methylmethanaminehydrochloride (22) (0.4 g, 1.19 mmol) and urea (23) (0.11 g, 1.83 mmol)in water (10 ml) was refluxed overnight. At this time, crystalsseparated from the liquid and were then collected by filtration. Theprecipitated product (24) was washed with water and dried. Yield 0.3 g,72%. MS, m/z 340.22 (M+H⁺).

Example 7. Synthesis of(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-N-methylsulfuricdiamide (26)

Preparation of(1-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-1-N-methylsulfuricdiamide (26)

A solution of1-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-N-methylmethanaminehydrochloride (22) (0.2 g, 0.6 mmol), sulfamide (25) (0.16 g, 1.68 mmol)in dioxane (15 ml) was heated to reflux for 6 hours. The reactionmixture was then cooled, and the solvent was evaporated. The residue wasdissolved in water (10 ml), and the aqueous was extracted with ethylacetate three times. The combined ethyl acetate layers were combined,dried, and evaporated. The residue (26) was purified by automated columnchromatography using petroleum ether and ethyl acetate as eluents. Yield70%. MS, m/z 376.28 (M+H⁺).

Example 8. Synthesis of4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-amine (28)

Preparation of 4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-amine (28)

To a solution of 3,5-bis(trifluoromethyl)benzene-1,2-diamine (1) (0.40g, 1.6 mmol) in acetonitrile (15 mL) was added a solution of cyanogenbromide in acetonitrile (27) (5 M, 0.66 mL, 3.3 mmol). The resultantmixture was allowed to stir at room temperature for 23 hours. At thistime, the reaction was concentrated in vacuo, and the crude product waspurified by automated flash chromatography to afford the (28); confirmedby LCMS (positive ion mode)).

Example 9. Synthesis of2-((methylsulfonyl)methyl)-4,6-bis(trfluoromethyl)-1H-benzo[d]imidazole(32)

Preparation ofN-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-(methylthio)acetamide (30)

To a mixture of 3,5-bis(trifluoromethyl)benzene-1,2-diamine (1) (0.50 g,2.1 mmol), 2-(methylthio)acetic acid (29) (0.22 g, 2.1 mmol) and HATU(1.01 g, 2.67 mmol) in dichloromethane (15 mL) was added triethylamine(0.9 mL, 6 mmol). The resultant solution was stirred at room temperaturefor 24 hours. The reaction was then washed with a saturated aqueoussolution of sodium bicarbonate (2×30 mL). The organic phase was thendried over anhydrous sodium sulfate and concentrated in vacuo. The crudeproduct was purified by automated flash chromatography (2:1 hexane:ethylacetate) to afford the title compound (30) (0.40 g, 59%; confirmed byLCMS (positive ion mode).) as a pale yellow oil, which crystallized onstanding. The other regioisomer was not observed during the course ofpurification.

Preparation of2-((methylthio)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole(31)

To a solution ofN-(2-amino-3,5-bis(trifluoromethyl)phenyl)-2-(methylthio)acetamide (30)(0.40 g, 1.2 mmol) in tetrahydrofuran (3 mL) was added glacial aceticacid (2 mL). The reaction was scaled in a microwave reaction vial andheated at 130° C. for 30 minutes. The resultant solution wasconcentrated in vacuo, taken up in ethyl acetate (40 mL), and washedwith a saturated aqueous solution of sodium bicarbonate (2×10 mL). Theorganic phase was then dried over anhydrous sodium sulfate andconcentrated in vacuo. The crude product was purified by automated flashchromatography (2:1 hexane:ethyl acetate) to afford the title compound(31) (0.34 g, 89%; confirmed by LCMS (positive ion mode)) as a yellowsolid.

Preparation of2-((methylsulfonyl)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole(32)

To a solution of2-((methylthio)methyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole(31) (0.34 g, 1.1 mmol) in dichloromethane (20 mL) was added m-CPBA(77%, 0.73 g, 3.2 mmol) and sodium bicarbonate (0.45 g, 5.4 mmol). Theresultant mixture was stirred at room temperature for 17 hours, at whichtime dichloromethane was added (30 mL). To the resultant solution wasadded an aqueous solution of sodium hydroxide (2 M, 10 mL) and asaturated aqueous solution of sodium thiosulfate (10 mL). The mixturewas allowed to stir for 1 hours. The aqueous layer was subsequentlyseparated, acidified with hydrochloric acid (2 M, 12 mL), extracted withethyl acetate (3×30 mL), and dried over anhydrous sodium sulfate. Theresultant oil was taken up in an ethyl acetate:methanol mixture (1:1)and filtered. The filtrate was then concentrated in vacuo, and theproduct was purified by automated flash chromatography to afford thetitle compound (32) (confirmed by LCMS (positive ion mode)).

Example 10. Synthesis ofN-((5,7-bis(trfluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)methanesulfonamide(34)

Preparation of N-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)methanesulfonamide (34)

To a solution of(5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methanamine hydrogenchloride (5) (0.30 g, 0.94 mmol) and DIPEA (0.3 mL, 2 mmol) indichloromethane (25 mL) at 0° C. was added methanesulfonyl chloride (33)(0.07 mL, 0.94 mmol) dropwise via syringe. The resultant solution waswarmed to room temperature, stirred for 72 hours, and then concentratedin vacuo. The resultant oil was then taken up in dichloromethane (30 mL)and washed with a saturated aqueous solution of sodium bicarbonate (20mL) and brine (20 mL). The organic layer was dried over anhydrous sodiumsulfate and concentrated in vacuo. The crude product was purified byautomated flash chromatography (ethyl acetate) to afford the titlecompound (34) (confirmed by LCMS (positive ion mode).

Example 11. Synthesis of(R)-2-(pyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole(39)

Preparation of(R)-2-(pyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-H-benzo[d]imidazole(39) 3,5-bis(trifluoromethyl)benzene-1,2-diamine (1) (1.5 g, 6.14 mmol),(R)-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (35) (1.30 g,6.04 mmol), TEA (1.1 mL, 7.98 mmol), and HATU (3.2 g, 8.42 mmol) weredissolved in DCM (15 mL). This solution was stirred overnight at roomtemperature and then concentrated in vacuo. The residue was taken up inethyl acetate (50 mL) and then washed sequentially with saturatedaqueous ammonium chloride (20 mL), saturated sodium bicarbonate (20 mL),and brine (20 mL). The organic fraction was then dried over anhydroussodium sulfate, filtered, and concentrated in vacuo. The residue waspurified by automated column chromatography (smooth gradient 20→70%ethyl acetate:petroleum ether) to afford a mixture of the desiredisomers (36) and (37), confirmed by LCMS (positive ion mode).

The mixture of isomers was taken up in THF/AcOH (95/5) and heated usinga microwave at 140° C. for 2 hours. The reaction was concentrated invacuo, taken up in EtOAc, and then washed with water (30 mL), saturatedaqueous sodium bicarbonate (30 mL), and brine (30 mL). The organic layerwas dried over anhydrous sodium sulfate then concentrated in vacuo. Theresidue was purified by automated column chromatography (smooth gradient20→70% ethyl acetate:petroleum ether), and this initial purification wasfollowed by a second round of automated column chromatography (smoothgradient 0→50% ethyl acetate:dichloromethane) to afford theN-Boc-protected product (38) as clear colorless gum. The product wastaken up in HCl saturated ethyl acetate and stirred for two hours atroom temperature. The clear mixture turned milky over time and wascondensed in vacuo to give the HCl salt of the product (39) as a whitesolid (1.00 g, 51% over two steps). ¹H NMR (300 MHz, CD₃OD) δ 2.24-2.40(m, 3H), 2.67-2.75 (m, 1H), 3.52-3.59 (m, 1H), 3.63-3.70 (m, 1H), 5.17(t, J=6 Hz, 1H), 7.86 (s, 1H), 8.25 (s, 1H).

Example 12. Synthesis of2-(3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)-N-(1-methylcyclobutyl)acetamide(42)

Preparation of2-(3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)-N-(1-methylcyclobutyl)acetamide(42)

To a solution of2-(azetidin-3-ylmethyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole(40) (0.81 g, 2.5 mmol) and triethylamine (1.8 mL, 13 mmol) inacetonitrile (50 mL) was added 2-chloro-N-(1-methylcyclobutyl)acetamide(41) (0.41 g, 2.5 mmol). The resultant solution was stirred at 40° C.for 17 hours and concentrated in vacuo. The crude product was purifiedby automated flash chromatography to afford the title compound (42). Theproduct was confirmed by LCMS (positive ion mode).

Example 13. Synthesis of1-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin2-one hydrochloride (47)

Synthesis of tert-butyl4-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)-3-oxopiperazine-1-carboxylate(45)

2-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)acetic acid (2) (2.0 g,7.77 mmol), 3,5 bis(trifluoromethyl)-O-phenylenediamine (44) (2.15 g,8.81 mmol), HOBt (1.36 g, 10.1 mmol), EDC (1.93 g, 10.1 mmol), anddiisopropylethylamine (2.3 mL, 13.2 mmol) were stirred in DMF (15 mL) atroom temperature for 14 hours. The residue was concentrated in vacuo andwas taken up in EtOAc (200 ml). The reaction mixture was washedsequentially with NH₄C1 (saturated solution), NaHCO₃ (saturatedsolution), and brine. The organics were separated, dried (Na₂SO₄) andconcentrated in vacuo. The residue was purified by automated columnchromatography (EtOAc/PE) to give tert-butyl4-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)-3-oxopiperazine-1-carboxylate(45) (3.5 g, 93%; confirmed by LCMS (positive ion mode)).

Synthesis of tert-butyl4-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopiperazine-1-carboxylate(46)

tert-Butyl4-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)-3-oxopiperazine-1-carboxylate(45) (3.5 g, 7.23 mmol) was heated in THF/AcOH (1:1, 20 mL) using amicrowave at 140° C. for 45 minutes. The residue was concentrated invacuo, and the residue was then taken up in EtOAc (150 ml). The reactionmixture was washed sequentially with NaHCO₃ (saturated solution) andbrine. The organics were separated, dried (Na₂SO₄), and concentrated invacuo. The residue was purified by automated column chromatography(EtOAc) to give tert-butyl4-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopiperazine-1-carboxylate(46) (3.1 g, 92%; confirmed by LCMS (positive ion mode))

Synthesis of1-((5,7-bis(trfluoromethyl)-H-benzo[d]imidazol-2-yl)methyl)piperazin-2-onehydrochloride (47)

tert-butyl4-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopiperazine-1-carboxylate(46) (3.1 g, 6.65 mmol) was taken up in EtOAc, and the solution wasflushed with HCl (g) for 5 minutes. The resultant suspension was stirredat room temperature for 30 minutes then concentrated in vacuo. Theresultant solid was dried under high vacuum for 14 hours to give1-((5,7-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2-onehydrochloride (47) in a quantitative manner. ¹H NMR (300 mHz, CD₃OD) δ3.70-3.75 (m, 2H), 4.00-4.10 (m, 4H), 5.10 (s, 1H), 8.15 (s, 1H), 8.2(s, 1H), 7.86 (s, 1H).

Example 14. Synthesis of2-(azetidin-3-ylmethyl)-4,6-bis(trfluoromethyl)-1H-benzo[d]imidazole(51)

Preparation of tert-butyl3-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)azetidine-1-carboxylate(49)

To a mixture of 3,5-bis(trifluoromethyl)benzene-1,2-diamine (43) (3.00g, 12.3 mmol), 2-(1-(tert-butoxycarbonyl)azetidin-3-yl)acetic acid (48)(2.64 g, 12.3 mmol), and HATU (6.08 g, 16.0 mmol) in dichloromethane (60mL) was added triethylamine (5.1 mL, 36.9 mmol). The resultant solutionwas stirred at room temperature for 23 hours and then washed with asaturated aqueous solution of sodium bicarbonate (2×70 mL). The organicphase was then dried over anhydrous sodium sulfate and concentrated invacuo. The crude product was purified by automated flash chromatography(1:1 hexane:ethyl acetate) to afford the title compound (49) (3.46 g,64%) as a white foam. The other regioisomer was not observed during thecourse of purification. The product was confirmed by LCMS (positive ionmode).

Preparation of tert-butyl3-((4,6-bis(trfluoromethyl)-H-benzo[d]imidazol-2-yl)methyl)azetidine-1-carboxylate(50)

To a solution of tert-butyl3-(2-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-2-oxoethyl)azetidine-1-carboxylate(49) (2.54 g, 5.75 mmol) in tetrahydrofuran (15 mL) was added glacialacetic acid (3 mL). The reaction was sealed in a microwave reaction vialand reacted at 130° C. for 45 minutes. The resultant solution wasconcentrated in vacuo, taken up in ethyl acetate (80 mL), and washedwith a saturated aqueous solution of sodium bicarbonate (20 mL). Theorganic phase was then dried over anhydrous sodium sulfate andconcentrated in vacuo. The crude product was purified by automated flashchromatography (1:1 hexane:ethyl acetate) to afford the title compound(50) (2.12 g, 87%) as a white solid. The product was confirmed by LCMS(positive ion mode).

Preparation of2-(azetidin-3-ylmethyl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole(51)

To a solution of tert-butyl3-((4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)azetidine-1-carboxylate(50) (1.06 g, 2.50 mmol) in dichloromethane (40 mL) was addedtrifluoroacetic acid (2.4 mL, 31 mmol). The resultant solution wasallowed to stir at room temperature for 2.5 hours and was thenconcentrated in vacuo. The resultant oil was taken up in toluene (30 mL)then concentrated in vacuo. The crude product was purified by automatedflash chromatography (80:20:1 dichloromethane:methanol:ammoniumhydroxide) to afford the title compound (51) as a cream colored foam; ¹HNMR (400 MHz, CD₃OD) δ 3.22 (quintet, 1H), 3.28 (d, 2H), 3.47 (septet,1H), 3.99 (m, 2H), 4.16 (m, 2H), 7.64 (s, 1H), 8.00 (s, 1H). The productwas confirmed by LCMS (positive ion mode).

Example 15. Synthesis of2-(2-oxopiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)acetamide(54)

Synthesis of tert-butyl3-oxo-4-(2-oxo-2-((5-(trifluoromethyl)pyridin-2-yl)amino)ethyl)piperazine-1-carboxylate(53)

To a solution of 5-(trifluoromethyl)pyridin-2-amine (52) (0.162 g, 1mmol), 2-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)acetic acid (44)(0.257 g, 1 mmol), and HATU (0.46 g, 1.3 mmol) in DMF 3 ml was addedtriethylamine (0.3 ml, 3 mmol). The mixture was heated in a microwave at75° C. for 2 hours. The reaction mixture was diluted with ethyl acetateand washed with saturated sodium bicarbonate aqueous solution and brine.The organic layer was dried over sodium sulfate and concentrated. Theresidue was purified by automated flash chromatography using pet etherand ethyl acetate as eluents. Yield 0.15 g, 50%. LCMS m/z 401.9 (M+H⁺).

Synthesis of2-(2-oxopiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)acetamide(54)

To a solution of tert-butyl3-oxo-4-(2-oxo-2-((5-(trifluoromethyl)pyridin-2-yl)amino)ethyl)piperazine-1-carboxylate(53) (0.15 g, 0.5 mmol) in ethyl acetate (3 ml) was added saturated HClsolution in ethyl acetate (2 ml). The reaction mixture was stirred atroom temperature for 30 minutes. The reaction mixture was thenconcentrated and dried in vacuo to give2-(2-oxopiperazin-1-yl)-N-(5-(trifluoromethyl)pyridin-2-yl)acetamide(54) as HCl salt. Yield 0.165 g, 98%. LCMS m/z 301.9 (M+H⁺).

Example 16. Synthesis of1-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-one(61)

Synthesis of tert-butyl4-(3-ethoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate (57)

tert-Butyl 3-oxopiperazine-1-carboxylate (55) (4.0 g, 20 mmol) wasstirred in dry DMF at room temperature under Ar. NaH (60% dispersion inmineral oil) (960 mg, 24 mmol) was added, and the reaction stirred for30 minutes. Ethyl bromopropionate (56) (2.55 mL, 20 mmol) was added inone portion, and stirring continued for 14 hours. The reaction waspartitioned between EtOAc and H₂O. The organics were separated, dried(Na₂SO₄), and concentrated in vacuo to give tert-butyl4-(3-ethoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate (57) as a cruderesidue, which was used in the subsequent reaction without additionalpurification. The product was confirmed with LCMS (positive ion mode).

Synthesis of 3-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)propanoicacid (58)

tert-Butyl 4-(3-ethoxy-3-oxopropyl)-3-oxopiperazine-1-carboxylate (57);as the crude residue from the previous step) and LiOH.H₂O (1.26 g, 30mmol) were stirred in THF/H₂O/MeOH (40/40/15 mL) at room temperature for16 hours. The resultant solution was filtered to remove solidprecipitation. The organic solvent was removed in vacuo, and thesolution was acidified with IM HCl. The reaction was extracted withEtOAc (3×75 mL), and the organics were dried (Na₂SO₄) and concentratedin vacuo to give3-(4-(tert-butoxycarbonyl)-2-oxopiperazin-1-yl)propanoic acid (58) (4.4g, 81% from tert-Butyl 3-oxopiperazine-1-carboxylate (55). The productwas confirmed by LCMS (negative ion mode).

Synthesis of ten-butyl4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropyl)-3-oxopiperazine-1-carboxylate(59)

3-(4-(tert-Butoxycarbonyl)-2-oxopiperazin-1-yl)propanoic acid (58) (1.52g, 5.6 mmol), 3,5 bis(trifluoromethyl)-O-phenylenediamine (1) (1.36 g,5.6 mmol), HATU (2.91 g, 7.84 mmol) and TEA (1.56 mL, 11.8 mmol) werestirred in DCM (50 mL) at room temperature for 14 hours. The reactionwas diluted with DCM (100 mL), washed sequentially with NH₄Cl (saturatedsolution), NaHCO₃ (saturated solution) and brine, the organicsseparated, dried (Na₂SO₄), and concentrated in vacuo. The residue waspurified by automated column chromatography (100% EtOAc/PE) to givetert-butyl4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropyl)-3-oxopiperazine-1-carboxylate(59) (2.64 g, 95%; confirmed by LCMS (positive ion mode)).

Synthesis of1-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-onehydrochloride (61)

tert-Butyl4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropyl)-3-oxopiperazine-1-carboxylate(59) (500 mg, 1.0 mmol) was heated in THF/AcOH (95:5, 2 mL) using amicrowave at 140° C. for 2.5 hours. The residue was concentrated invacuo to give crude tert-butyl4-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)-3-oxopiperazine-1-carboxylate(60). The crude material was taken up in EtOAc, and the solution flushedwith HCl (g) for 5 minutes. The suspension was stirred at roomtemperature for 20 minutes and concentrated in vacuo. The residue waspurified by reverse phase HPLC to give1-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-onehydrochloride (61).

Example 17. Synthesis of2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1-(piperazin-1-yl)ethanone(68)

Synthesis of tert-butyl4-(3-ethoxy-3-oxopropanoyl)piperazin-1-carboxylate (64)

tert-Butyl piperazine-1-carboxylate (62) (1 g, 5.4 mmol) and TEA (837μL, 6.0 mmol) were stirred in DCM at room temperature. Ethyl malonylchloride (63) (810 μL, 5.4 mmol) was added in one portion, and thereaction stirred at room temperature for 1 hour. The reaction wasdiluted with DCM and washed sequentially with NH₄Cl (saturated solution)and NaHCO₃ (saturated solution). The organics were separated, dried(Na₂SO₄), and concentrated in vacuo. The residue was purified byautomated column chromatography (100% EtOAc) to give tert-butyl4-(3-ethoxy-3-oxopropanoyl)piperazine-1-carboxylate (64) (1.24 g, 77%).The product was confirmed by LCMS (positive ion mode).

Synthesis of 3-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-oxopropanoicacid (65)

tert-Butyl 4-(3-ethoxy-3-oxopropanoyl)piperazine-1-carboxylate (64)(1.24 g, 2.8 mmol) and LiOH.H₂O (176 mg, 4.2 mmol) was stirred inTHF/H₂O/MeOH (30/30/10 mL) at room temperature for 14 hours. The organicsolvents were removed in vacuo, and the solution was acidified with 1 MHCl and extracted with EtOAc. The organics were separated, dried(Na₂SO₄), and concentrated in vacuo to give3-(4-(tert-butoxycarbonyl)piperazin-1-yl)-3-oxopropanoic acid (65) in aquantitative fashion. The product was confirmed by LCMS (negative ionmode) and was used in the subsequent reaction without additionalpurification

Synthesis of tert-butyl4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropanoyl)piperazine-1-carboxylate(66)

3-(4-(tert-Butoxycarbonyl)piperazin-1-yl)-3-oxopropanoic acid (65) (761mg, 2.8 mmol), 3,5-bis(trifluoromethyl)benzene-1,2-diamine (43) (744 mg, 3.05 mmol), HATU (1.6 g, 4.27 mmol), and TEA (906 μL, 6.5 mmol) werestirred in DCM at room temperature for 14 hours. The precipitate wasremoved by filtration. The filtrate was diluted with DCM and washedsequentially with NH₄Cl (saturated solution) and NaHCO₃ (saturatedsolution). The organics were separated, dried (Na₂SO₄), and concentratedin vacuo. The residue was purified by automated column chromatography(EtOAc/PE, 50/50) to give tert-butyl4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropanoyl)piperazine-1-carboxylate(66) (650 mg, 42%). The product was confirmed by LCMS (positive ionmode).

Synthesis of2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1-(piperazin-1-yl)ethanonehydrochloride (68)

tert-Butyl4-(3-((2-amino-3,5-bis(trifluoromethyl)phenyl)amino)-3-oxopropanoyl)piperazine-1-carboxylate(66) (650 mg, 1.31 mmol) was heated in THF/AcOH (95:5, 2 mL) using amicrowave at 140° C. for 2.5 hours. The residue was concentrated invacuo to give crude tert-butyl4-(2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)acetyl)piperazine-1-carboxylate(67). The crude was taken up in EtOAc, and the solution was flushed withHCl (g) for 5 minutes. The suspension was stirred at room temperaturefor 20 minutes and concentrated in vacuo. The residue was purified byreverse phase HPLC to give2-(4,6-bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1-(piperazin-1-yl)ethanonehydrochloride (68).

Example 18. Synthesis of1-((1-(2-hydroxy-2-methylpropyl)-4,6-bis(trfluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2-one(30)

Preparation of1-((1-(2-hydroxy-2-methylpropyl)-4,6-bis(trifluromethyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2-one(72)

tert-Butyl-4-((5,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopiperazine-1-carboxylate(69) (0.50 g, 1.25 mmol) and N-(tert-butyl)-2-chloroacetamide (70) (0.20g, 1.3 mmol) were dissolved in acetone (10 mL). The resultant solutionwas stirred at reflux for 48 hours, cooled to room temperature, andpoured into water (60 mL). The aqueous mixture was extracted with ethylacetate (3×50 mL). The combined organic fractions were dried withanhydrous sodium sulfate, filtered, then condensed in vacuo to give theBoc-protected product (71) (0.60 g, 93%), which was confirmed by LCMS(positive ion mode). A portion of this material (100 mg, 0.20 mmol) wastaken up into ethyl acetate (20 mL). HCl gas was bubbled through thissolution for one minute. The reaction was then stirred at roomtemperature for 30 minutes and concentrated in vacuo. The resultingresidue was purified by automated flash chromatography to afford thedesired free amine (72). The product was confirmed by LCMS (positive ionmode).

Example 19. Synthesis of1-((4,6-dichloro-1-(2-hydroxy-2-methylpropyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2-one(33)

Preparation of1-((4,6-dichloro-1-(2-hydroxy-2-methylpropyl)-1H-benzo[d]imidazol-2-yl)methyl)piperazin-2-one(75)

tert-Butyl-4-((4,6-dichloro-1H-benzo[d]imidazol-2-yl)methyl)-3-oxopiperazine-1-carboxylate(300 mg, 0.75 mmol) (69), 2,2-dimethyloxirane (73) (0.67 ml, 542 mg, 7.5mmol), and potassium carbonate (1.04 g, 7.5 mmol) were taken up inacetone (5 mL). The reaction was heated at 110° C. by microwaveirradiation for 1 hour. The solution was concentrated in vacuo, and theresidue containing the Boc-protected product (74) was taken up in ethylacetate. HCl gas was bubbled through this solution for 1 minute. Thesolution was then stirred for 30 minutes at room temperature andconcentrated in vacuo. The resulting residue was purified by automatedflash chromatography to afford the desired free amine (75). The productwas confirmed by LCMS (positive ion mode).

Following the general procedures as set forth in exemplary syntheticprocedures above, the following compounds listed in Table 1 wereprepared. Mass spectrometry was employed with final compounds and atvarious stages throughout the synthesis as a confirmation of theidentity of the product obtained (M+1). For the mass spectrometricanalysis, samples were prepared at an approximate concentration of 1μg/mL in acetonitrile with 0.1% formic acid. Samples were manuallyinfused into an Applies Biosystems API3000 triple quadrupole massspectrometer and scanned in Q1 in the range of 50 to 700 m/z.

TABLE 1 No. Structure Mol. Wt. Chemical Name  1

323.237 2-(azetidin-3-ylmethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  2

256.131 2-(azetidin-3-ylmethyl)-5,6- dichloro-1H-benzo[d]imidazole  3

436.395 2-(2-(azetidin-3-ylmethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)-N-(tert- butyl)acetamide  4

273.254 2-(azetidin-3-yl)-N-(2,2,2- trifluoro-1-(52yridine-2-yl)ethyl)acetamide  5

259.228 2-(azetidin-3-yl)-N-(5- (trifluoromethyl)52yridine-2-yl)acetamide  6

369.289 2-(2-(azetidin-3-ylmethyl)-5,6- dichloro-1H-benzo[d]imidazol-1-yl)-N-(tert-butyl)acetamide  7

273.254 2-(azetidin-3-yl)-N-((6- (trifluoromethyl)52yridine-3-yl)methyl)acetamide  8

328.237 1-(2-(azetidin-3-ylmethyl)-5,6- dichloro-1H-benzo[d]imidazol-1-yl)-2-methylpropan-2-ol  9

395.343 1-(2-(azetidin-3-ylmethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-1-yl)-2- methylpropan-2-ol  10

323.237 2-(azetidin-3-ylmethyl)-5,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  11

365.274 1-(3-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)azetidin-1- yl)ethanone  12

380.288 2-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)azetidin-1- yl)ethanone  13

394.315 3-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)propan- 1-one  14

422.368 3-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)-3- methylbutan-1-one  15

408.341 4-amino-1-(3-((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)butan 1-one  16

326.238 N-(3,5- bis(trifluoromethyl)benzy]) azetidine-3-carboxamide  17

340.264 (S)-N-(3,5- bis(trifluoromethyl)benzyl)pyrrolidine-2-carboxamide  18

340.264 ®-N-(3,5- bis(trifluoromethyl)benzyl) pyrrolidine-2-carboxamide 19

436.395 2-(3-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)-N- (tert-butyl)acetamide  20

448.405 2-(3-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)azetidin-1-yl)-N-(1- methylcyclobutyl)acetamide  21

216.067 (5,6-dichloro-1H- benzo[d]imidazol-2- yl)methanamine  22

273.119 2-amino-N-((5,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)acetamide  23

283.173 (5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)methanamine 24

340.224 2-amino-N-((5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)acetamide  25

311.226 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)propan-2-amine  26

309.21 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclopropanamine  27

325.253 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-2-methylpropan-1-amine  28

325.21 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)acetamide  29

338.766 6-(3-chloro-4-fluorophenyl)-2- (pyrimidin-5-ylmethyl)-1H-benzo[d]imidazole  30

384.355 2-(pyrimidin-5-ylmethyl)-6-(4- (2,2,2-trifluoroethoxy)phenyl)-1H-benzo[d]imidazole  31

367.333 (S)-2-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-4-methylpentan-1- amine  32

383.289 tert-butyl ((4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)carbamate  33

297.2 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)ethanamine 34

325.253 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-2-methylpropan-2-amine  35

311.226 3-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)propan-1-amine  36

368.278 2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- 2-methylpropanamide  37

382.304 3-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- 2,2-dimethylpropanamide  38

283.173 (5,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)methanamine 39

340.224 2-amino-N-((5,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)acetamide  40

397.315 tert-butyl ((5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)(methyl)carbamate  41

297.2 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N-methylmethanamine  42

354.251 2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- N-methylacetamide  43

368.278 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-N-methyl-2- (methylamino)acetamide  44

382.304 2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- N,2-dimethylpropanamide  45

325.253 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-N-methylethanamine  46

339.236 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-N-methylacetamide  47

327.226 (S)-1-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)-2-methoxyethanamine  48

410.357 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-2-(tert-butylamino)- N-methylacetamide  49

311.226 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N,N-dimethylmethanamine  50

340.224 1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-1-methylurea  51

323.237 (S)-2-(pyrrolidin-2-yl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  52

323.237 (R)-2-(pyrrolidin-2-yl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  53

309.21 2-(azetidin-3-yl)-4,6- bis(trifluoromethyl)-1H- benzo[d]imidazole 54

323.237 (1r,3r)-3-(4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclobutanamine  55

323.237 (1s,3s)-3-(4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclobutanamine  56

376.278 N-{[4,6-bis(trifluoromethyl)- 1H-benzimidazol-2-yl]methyl}-N-methylsulfuric diamide  57

375.29 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-N- methylmethanesulfonamide  58

455.355 (R)-2-amino-N-(2-(((4,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)(methyl)amino)-2- oxoethyl)-3-methoxypropanamide  59

346.231 2-(pyrimidin-5-ylmethyl)-5,7- bis(trifluoromethyl)-1H-benzo[d]imidazole  60

367.29 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)pivalamide  61

381.316 N-((5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)-3,3- dimethylbutanamide  62

382.304 1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-3-(tert-butyl)urea  63

298.184 2-(methoxymethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  64

312.211 2-(2-methoxyethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  65

336.156 2-(2,2,2-trifluoroethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  66

346.249 2-((methylsulfonyl)methyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  67

332.204 2-(pyrazin-2-yl)-4,6- bis(trifluoromethyl)-1H- benzo[d]imidazole 68

346.231 2-(pyrazin-2-ylmethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  69

269.147 4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-amine  70

284.158 (5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2- yl)methanol 71

345.242 2-(pyridin-3-ylmethyl)-5,7- bis(trifluoromethyl)-1H-benzo[d]imidazole  72

345.242 2-(pyridin-4-ylmethyl)-5,7- bis(trifluoromethyl)-1H-benzo[d]imidazole  73

424.384 (S)-3-(aminomethyl)-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- 5-methylhexanamide  74

230.094 2-(5,7-dichloro-1H- benzo[d]imidazol-2- yl)ethanamine  75

339.236 (3R,5S)-5-(5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)pyrrolidin-3-ol  76

311.226 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N-methylethanamine  77

383.289 (5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyltert-butylcarbamate  78

391.289 (5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyldimethylsulfamate  79

321.297 (5-(4-(2,2,2- trifluoroethoxy)phenyl)-1H- benzo[d]imidazol-2-yl)methanamine  80

275.709 (5-(3-chloro-4-fluorophenyl)- 1H-benzo[d]imidazol-2-yl)methanamine  81

361.263 N-((4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)methyl)methanesulfonamide  82

351.29 1-((4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)cyclopentanamine  83

341.275 (R)-4-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)thiazolidine  84

341.275 (R)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)thiazolidine  85

341.275 (S)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)thiazolidine  86

373.274 (R)-4-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)thiazolidine 1,1-dioxide  87

373.274 (R)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)thiazolidine 1,1-dioxide  88

373.274 (S)-2-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)thiazolidine 1,1-dioxide  89

341.227 2-((2S,4R)-4-fluoropyrrolidin- 2-yl)-4,6-bis(trifluoromethyl)-1H-benzo[d]imidazole  90

341.227 2-((2S,4S)-4-fluoropyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-1H- benzo[d]imidazole  91

359.218 (S)-2-(4,4-difluoropyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-1H- benzo[d]imidazole  92

353.263 2-((2R,4R)-4- methoxypyrrolidin-2-yl)-4,6-bis(trifluoromethyl)-1H- benzo[d]imidazole  93

323.237 (R)-2-(pyrrolidin-3-yl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole  94

339.279 (S)-1-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)-N,2-dimethylpropan-1-amine  95

353.306 (S)-1-(4,6-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)-N,3-dimethylbutan-1-amine  96

353.306 (1S,2S)-1-(4,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-N,2- dimethylbutan-1-amine  97

197.185 2-(4,6-difluoro-1H- benzo[d]imidazol-2- yl)ethanamine  98

223.222 (S)-4,6-difluoro-2-(pyrrolidin-2- yl)-1H-benzo[d]imidazole  99

223.222 (S)-5,6-difluoro-2-(pyrrolidin-2- yl)-1H-benzo[d]imidazole 100

223.222 (S)-4,5-difluoro-2-(pyrrolidin-2- yl)-1H-benzo[d]imidazole 101

241.212 (S)-4,5,6-trifluoro-2- (pyrrolidin-2-yl)-1H- benzo[d]imidazole102

320.19 2-(1H-pyrazol-4-yl)-5,7- bis(trifluoromethyl)-1H-benzo[d]imidazole 103

348.25 2-(3,5-dimethyl-1H-pyrazol-4- yl)-5,7-bis(trifluoromethyl)-1H-benzo[d]imidazole 104

320.19 2-(1H-pyrazol-3-yl)-5,7- bis(trifluoromethyl)-1H-benzo[d]imidazole 105

360.26 2-(3-cyclopropyl-1H-pyrazol-5- yl)-5,7-bis(trifluoromethyl)-1H-benzo[d]imidazole 106

388.19 5,7-bis(trifluoromethyl)-2-(3- (trifluoromethyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole 107

319.21 2-(1H-pyrrol-3-yl)-5,7- bis(trifluoromethyl)-1H-benzo[d]imidazole 108

299.156 1-((5,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 109

298.264 1-((5-(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 110

366.262 1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 111

394.315 1-((4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-3,3-dimethylpiperazin-2-one 112

299.156 1-((4,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 113

302.252 2-(2-oxopiperazin-1-yl)-N-(4- (trifluoromethyl)66yridine-2-yl)acetamide 114

302.252 2-(2-oxopiperazin-1-yl)-N-(5- (trifluoromethyl)66yridine-2-yl)acetamide 115

316.279 N-(2-methyl-6- (trifluoromethyl)66yridine-3-yl)-2-(2-oxopiperazin-1- yl)acetamide 116

316.279 2-(2-oxopiperazin-1-yl)-N-((6- (trifluoromethyl)67yridine-3-yl)methyl)acetamide 117

305.256 N-(1-methyl-3- (trifluoromethyl)-1H-pyrazol-5-yl)-2-(2-oxopiperazin-1- yl)acetamide 118

287.281 2-(piperidin-4-yl)-N-(5- (trifluoromethyl)67yridine-2-yl)acetamide 119

299.156 4-((5,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 120

300.141 4-((5,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)morpholin-3-one 121

313.182 1-(2-(5,6-dichloro-1H- benzo[d]imidazol-2-yl)ethyl)piperazin-2-one 122

397.342 N-(tert-butyl)-2-(5,6-dichloro-2- (piperidin-4-ylmethyl)-1H-benzo[d]imidazol-1- yl)acetamide 123

356.29 1-(5,6-dichloro-2-(piperidin-4- ylmethyl)-1H-benzo[d]imidazol-1-yl)-2- methylpropan-2-ol 124

412.314 N-(tert-butyl)-2-(5,6-dichloro-2- ((2-oxopiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1- yl)acetamide 125

479.419 N-(tert-butyl)-2-(2-((2- oxopiperazin-1-yl)methyl)-4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-1- yl)acetamide 126

380.288 1-(2-(5,7-bis(trifluoromethyl)- 1H-benzo[d]imidazol-2-yl)ethyl)piperazin-2-one 127

367.246 4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)morpholin-3-one 128

367.246 4-((5,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)morpholin-3-one 129

438.367 1-((1-(2-hydroxy-2- methylpropyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)piperazin-2-one 130

344.332 2-(3,3-dimethyl-2-oxopiperazin- 1-yl)-N-(2,2,2-trifluoro-1-(69yridine-2-yl)ethyl)acetamide 131

316.279 2-(2-oxopiperazin-1-yl)-N- (2,2,2-trifluoro-1-(69yridine-2-yl)ethyl)acetamide 132

301.307 2-(piperidin-4-yl)-N-(2,2,2- trifluoro-1-(69yridine-2-yl)ethyl)acetamide 133

330.306 2-(3,3-dimethyl-2-oxopiperazin 1-yl)-N-(5-(trifluoromethyl)69yridine-2- yl)acetamide 134

440.367 N-(tert-butyl)-2-(5,6-dichloro-2 ((3,3-dimethyl-2-oxopiperazin-1-yl)methyl)-1H- benzo[d]imidamol-1- yl)acetamide 135

351.29 2-(piperidin-4-ylmethyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole 136

423.396 2-methyl-1-(2-(piperidin-4- ylmethyl)-4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-1-yl)propan- 2-ol 137

464.448 N-(tert-butyl)-2-(2-(piperidin-4 ylmethyl)-4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-1- yl)acetamide 138

507.472 N-(tert-butyl)-2-(2-((3,3- dimethyl-2-oxopiperazin-1-yl)methyl)-4,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-1-yl)acetamide 139

466.421 1-((1-(2-hydroxy-2- methylpropyl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- 3,3-dimethylpiperazin-2-one 140

301.307 2-(piperidin-4-yl)-N-((6- (trifluoromethyl)70yridine-3-yl)methyl)acetamide 141

344.332 (2-(3,3-dimethyl-2- oxopiperazin-1-yl)-N-((6-(trifluoromethyl)71yridine-3- yl)methyl)acetamide 142

351.29 2-(piperidin-4-ylmethyl)-5,6- bis(trifluoromethyl)-1H-benzo[d]imidazole 143

399.315 1-((5,6-dichloro-1-(2-hydroxy- 2-methylpropyl)-1H-benzo[d]imidazol-2-yl)methyl)- 3,3-dimethylpiperazin-2-one 144

371.262 1-((5,6-dichloro-1-(2-hydroxy- 2-methylpropyl)-1H-benzo[d]imidazol-2- yl)methyl)piperazin-2-one 145

371.262 1-((4,6-dichloro-1-(2-hydroxy- 2-methylpropyl)-1H-benzo[d]imidazol-2- yl)methyl)piperazin-2-one 146

438.367 1-((1-(2-hydroxy-2- methylpropyl)-5,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)piperazin-2-one 147

412.314 N-(tert-butyl)-2-(4,6-dichloro-2- ((2-oxopiperazin-1-yl)methyl)-1H-benzo[d]imidazol-1- (yl)acetamide 148

479.419 N-(tert-butyl)-2-(2-((2- oxopiperazin-1-yl)methyl)-5,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-1- yl)acetamide 149

366.262 1-((5,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 150

394.315 1-((5,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-3,3-dimethylpiperazin-2-one 151

466.421 1-((1-(2-hydroxy-2- methylpropyl)-5,6- bis(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)methyl)- 3,3-dimethylpiperazin-2-one 152

507.472 N-(tert-butyl)-2-(2-((3,3- dimethyl-2-oxopiperazin-1-yl)methyl)-5,6- bis(trifluoromethyl)-1H- benzo[d]imidazol-1-yl)acetamide 153

366.262 4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 154

327.209 1-((5,6-dichloro-1H- benzo[d]imidazol-2-yl)methyl)-3,3-dimethylpiperazin-2-one 155

380.288 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-piperazin-1-yl)ethanone 156

381.273 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-morpholinoethanone 157

379.3 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-(piperidin-1-yl)ethanone 158

394.315 2-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-1-(4-methylpiperazin-1-yl)ethanone 159

369.262 N-(3,5- bis(trifluoromethyl)benzyl)-2-oxopiperazine-1-carboxamide 160

302.228 1-((4,5,6,7-tetrafluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 161

284.237 1-((5,6,7-trifluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 162

284.237 1-((4,5,7-trifluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 163

266.247 1-((5,7-difluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 164

266.247 1-((4,7-difluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 165

266.247 1-((6,7-difluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 166

266.247 1-((5,6-difluoro-1H- benzo[d]imidazol-2-yl)methyl)piperazin-2-one 167

352.235 3-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)piperazin-2-one 168

300.2 2-amino-N-(3,5- bis(trifluoromethyl)benzyl) acetamide 169

354.291 N-(3,5- bis(trifluoromethyl)benzyl) piperidine-4-carboxamide 170

355.279 N-(3,5- bis(trifluoromethyl)benzyl) piperazine-1-carboxamide 171

365.317 1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)cyclohexanamine 172

408.341 (1S,2R)-2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)cyclohexane- carboxamide 173

408.341 (1R,2S)-2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)cyclohexane- carboxamide 174

408.341 (1R,2R)-2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)cyclohexane- carboxamide 175

408.341 (1S,2S)-2-amino-N-((5,7- bis(trifluoromethyl)-1H-benzo[d]imidazol-2- yl)methyl)cyclohexane- carboxamide 176

298.211 1-((5,7-dichloro-1H- benzo[d]imidazol-2-yl)methyl)cyclohexanamine 177

365.317 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N-methylcyclohexanamine 178

351.29 1-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclohexanamine 179

367.29 4-(5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)-N-methyltetrahydro-2H-pyran-4- amine 180

379.343 1-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-N-methylcyclohexanamine 181

367.29 4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)tetrahydro-2H-pyran- 4-amine 182

381.316 4-((5,7-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)methyl)-N-methyltetrahydro-2H-pyran- 4-amine 183

337.264 (R)-2-(piperidin-3-yl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole 184

337.264 (S)-2-(piperidin-3-yl)-4,6- bis(trifluoromethyl)-1H-benzo[d]imidazole 185

339.236 2-(4,6-bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)morpholine 186

351.29 (1R,2R)-2-(5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclohexanamine 187

351.29 (1S,2R)-2-(5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclohexanamine 188

351.29 (2S,2S)-2-(5,7- bis(trifluoromethyl)-1H- benzo[d]imidazol-2-yl)cyclohexanamine 189

340.264 (S)-N-(3,5- bis(trifluoromethyl)benzyl)pyrrolidine-2-carboxamide 190

340.264 (R)-N-(3,5- bis(trifluoromethyl)benzyl)pyrrolidine-2-carboxamide 191

290.257 (S)-N-(4-fluoro-3- (trifluoromethyl)benzyl)pyrrolidine-2-carboxamide 192

256.704 (S)-N-(3-chloro-4- fluorobenzyl)pyrrolidine-2- carboxamide 193

333.281 N-(4-fluoro-3- trifluoromethyl)benzyl)-2-(2-oxopiperazin-1-yl)acetamide 194

340.264 (R)-N-(pyrrolidin-2-ylmethyl)- 3,5-bis(trifluoromethyl)benzamide 195

340.264 (S)-N-(pyrrolidin-2-ylmethyl)- 3,5-bis(trifluoromethyl)benzamide 196

356.307 (S)-2-amino-N-(3,5- bis(trifluoromethyl)benzyl)-4-methylpentanamide 197

370.333 (S)-N-(3,5- bis(trifluoromethyl)benzyl)-4- (methyl-2-(methylamino)pentanamide 198

383.289 N-(3,5- bis(trifluoromethyl)benzyl)-2-(2-oxopiperazin-1-yl)acetamide 199

382.344 2-(1-aminocyclohexyl)-N-(3,5- bis(trifluoromethyl)benzyl)acetamide 200

416.36 (2R,5S)-N-(3,5- bis(trifluoromethyl)benzyl)-5-phenylpyrrolidine-2- carboxamide 201

314.227 3-amino-N-(3,5- bis(trifluoromethyl)benzyl) propanamide 202

328.253 N-(3,5- bis(trifluoromethyl)benzyl)-3- (methylamino)propanamide203

416.36 (2S,4R)-N-(3,5- bis(trifluoromethyl)benzyl)-4-phenylpyrrolidine-2- carboxamide 204

416.36 (2S,4S)-N-(3,5- bis(trifluoromethyl)benzyl)-4-phenylpyrrolidine-2- carboxamide 205

358.255 (2S,4S)-N-(3,5- bis(trifluoromethyl)benzyl)-4-fluoropyrrolidine-2- carboxamide 206

376.245 (S)-N-(3,5- bis(trifluoromethyl)benzyl)-4,4-difluoropyrrolidine-2- carboxamide 207

368.317 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)pyrrolidine-2- carboxamide 208

368.317 (S)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)pyrrolidine-2- carboxamide 209

447.396 (S)-N-(2-(3,5- bis(trifluoromethyl)phenyl) propan-2-yl)-1-sulfamoylpyrrolidine-2- carboxamide 210

354.291 (R)-N-(3,5- bis(trifluoromethyl)benzyl)-1- methylpyrrolidine-2-carboxamide 211

354.291 (S)-N-(3,5- bis(trifluoromethyl)benzyl)-1- methylpyrrolidine-2-carboxamide 212

382.344 (S)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)piperidine-2- carboxamide 213

384.317 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)morpholine-3- carboxamide 214

384.317 (S)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)morpholine-3- carboxamide 215

433.272 N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide 216

396.37 (1S,2R)-2-amino-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)cyclohexane- carboxamide 217

396.37 (1S,2S)-2-amino-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)cyclohexane- carboxamide 218

396.37 (1S,2S)-2-amino-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)cyclohexane- carboxamide 219

396.37 (S)-N-(1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)-4-methyl-2- (methylamino)pentanamide 220

366.301 (S)-N-(1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)pyrrolidine-2- carboxamide 221

366.301 (R)-N-(1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)pyrrolidine-2- carboxamide 222

384.292 (2R,4R)-N-(1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)-4-fluoro- pyrrolidine-2-carboxamide 223

384.292 (2R,4S)-N-(1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)-4-fluoro- pyrrolidine-2-carboxamide 224

300.2 2-amino-3-(3,5- bis(trifluoromethyl)phenyl) propanamide 225

431.256 N-(1-(3,5- bis(trifluoromethyl)phenyl) cyclopropyl)-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide 226

370.333 (R)-2-amino-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)-3-methyl- butanamide 227

384.36 (R)-2-amino-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)-4- methylpentanamide 228

386.333 (2R,3S)-2-amino-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)-3-methoxy- butanamide 229

398.386 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)-4-methyl-2- (methylamino)pentanamide 230

426.44 (R)-N-(2-(3,5-bis (trifluoromethyl)phenyl)propan-2-yl)-2-(isopropylamino)- 4-methylpentanamide 231

427.341 (R)-2-amino-N-(2-((1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)amino)-2-oxoethyl)- 3-methoxypropanamide 232

429.357 (R)-2-amino-N-(2-((2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)amino)-2-oxoethyl)- 3-methoxypropanamide 233

370.333 (R)-N-(3,5- bis(trifluoromethyl)benzyl)-4- methyl-2-(methylamino)pentanamide 234

396.37 (R)-N-(1-(3,5- bis(trifluoromethyl)phenyl)cyclopropyl)-4-methyl-2- (methylamino)pentanamide 235

356.307 (R)-N-(3,5- bis(trifluoromethyl)benzyl)-3- methyl-2-(methylamino)butanamide 236

382.348 (R)-N-(2-(3,5- bis(trifluoromethyl)phenyl)propan-2-yl)piperidine-2- carboxamide

Example 20. Ion Channel Studies

The generation of a HEK 293F cell line stably expressing human Na_(v)1.7was achieved by co-transfecting human SCN9A and human SCN1B cDNAs,subcloned into plasmid vectors and utilizing standard transfectiontechniques. Clones were selected using appropriate selection agents (0.3mg/mL Zeocin and 0.8 mg/mL Geneticin) and maintained in Dulbecco'sModified Eagle medium, 10% fetal bovine serum, 1% non essential aminoacids to ˜80% confluence at 37° C. in a humidified incubator with 95%atmosphere and 5% CO₂.

On the day of each experiment, cells that were grown to 80% confluencein a T75 flask were harvested for use on PatchXpress (Molecular Devices,CA, USA). Following a recovery period at 37° C. in a humidifiedincubator with 95% atmosphere and 5% CO₂ in Dulbecco's Modified EagleMedium, the media was replaced with an external recording solutioncontaining (in mM): 90 TEACl, 50 NaCl, 1.8 CaCl₂, 1 MgCl₂, 10 HEPES, 10glucose, adjusted to pH 7.4 with TEAOH and 300 mOsm with sucrose. Theinternal recording solution contained (in mM): 129 CsF, 2 MgCl₂, 11EGTA, 10 HEPES, 6 NaCl, 3 Na₂ATP adjusted to pH 7.2 with CsOH and 280mOsm with sucrose. The automated liquid handling facility of PatchXpressdispensed cells and added compound.

Modulation of Na_(v)1.7 channels by compounds was assessed by promotingthe channels into the inactivated state using a conditioning voltagepulse of variable amplitude, followed by a brief hyperpolarizing pulsewith a subsequent depolarized voltage step to measure the currentamplitude in the presence and absence of compound. Exemplary data areprovided in FIG. 1.

Example 21. Assays Modulation of Ion Channel Activity

The compounds described herein can also be assayed for modulation ofother voltage gated channels (e.g., other Na⁺ channel isoforms or Ca²⁺channels such as Ca_(v)3.2 T-type channels). Additional methods areknown in the art. Exemplary data obtained according to these methods arealso shown in FIG. 1.

Na_(v)1.5 Assay

Inhibition of the TTX-resistant Na_(v)1.5 sodium channel, a key cardiacion channel, can have profound effects on the duration and amplitude ofthe cardiac action potential and can result in arrhythmias and otherheart malfunctions. To assess the potential cardiac liability ofcompounds at an early stage in the drug discovery process, a Na_(v)1.5sodium channel screening assay was be performed on Molecular Device'sPatchXpress™ automated electrophysiology platform. Under voltage-clampconditions, Na_(v)1.5 currents were recorded from HEK cells expressingthe human Na_(v)1.5 channel in the absence and presence of increasingconcentrations of the test compound to obtain an IC₅₀ value. Theexternal recording solution contained (in mM): 90 TEACl, 50 NaCl, 1.8CaCl, 1 MgCl₂, 10 HEPES, 10 glucose, adjusted to pH 7.4 with TEA-OH andto 300 mOsm with sucrose (if necessary), while the internal patchpipette solution contained (in mM): 129 CsF, 2 MgCl₂, 11 EGTA, 10 HEPES,3 Na₂ATP adjusted to pH 7.2 with CsOH and to 290 mOsm with sucrose (ifnecessary). Na_(v) 1.5 channel currents were evoked using a cardiacaction potential waveform at 1 Hz, digitized at 31.25 kHz and low-passfiltered at 12 kHz.

Voltage-Gated Ca²⁺ Channels

The compounds described herein can also be studied as modulators ofvoltage-gated Ca²⁺ channels (e.g., Ca_(v)1.2, Ca_(v)2.2, Ca_(v)3.1, orCa_(v)3.2 channels). Exemplary methods are described herein.

A. Patch Clamp Methods

To record currents from Ca_(v)3.2 T-type Ca²⁺ channels expressed in HEKcells, the culture media can be replaced with extracellular solution(ECS) containing (in mM): 142 CsCl, 10 D-glucose, 2 CaCl₂, 1 MgCl₂, 10HEPES, pH adjusted to 7.4 with CsOH. Borosilicate glass patch pipettes,pulled on a P-97 micropipette puller (Sutter Instruments, Novato,Calif.) with typical resistances of 2-4 MW, can be backfilled withintracellular solution containing (in mM): 126.5 Cs-methanesulphonate, 2MgCl₂, 10 HEPES, 11 EGTA, 2 Na-ATP, pH adjusted to 7.3 CsOH. Voltageswere recorded in the whole-cell configuration at room temperature (˜21°C.) using an Axopatch 200B (Molecular Devices, Sunnyvale, Calif.)patch-clamp amplifier. Recordings can be low-pass filtered at 1 kHz (−3dB 4-pole Bessel filter), digitized at 2 kHz with a Digidata 1322Ainterface (Molecular Devices), and acquired using pClamp 9.2 (MolecularDevices), with no leak subtraction being used. Test compounds, preparedas 30 mM stock solutions in DMSO and diluted in extracellular buffer,can be applied through a gravity driven multi-barrelled array ofcapillaries (24 gauge) connected to reservoirs controlled by solenoidvalves. The effects of compounds on Ca_(v)3.2 slow and fast inactivationcan then be evaluated using different voltage protocols. The voltagedependence of fast and slow channel inactivation can be examined using atwo pulse protocol. Data were analyzed and fitted using OriginPro v.7.5(OriginLab, Northampton, Mass.) software.

B. High-Throughput Ca_(v)2.2/K_(ir)2.3 T-Type Fluorescent Assay

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysinecoated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior touse in the FLIPR assay. 100 μL of cells (1.4×10⁶ cell/mL) containingdoxycycline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression)were added to each well using a Multidrop (Thermo Scientific, Waltham,Mass.) and were maintained in 5% CO₂ incubator at 37° C. On the morningof the assay, cells were transferred to a 5% CO₂ incubator at 29° C.

Cells can then be washed with a wash buffer containing (in mM): 118NaCl, 18.4 HEPES, 11.7 D-glucose, 2 CaCl₂, 0.5 MgSO₄, 4.7 KCl, 1.2KH₂PO₄, pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescentindicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid(Sigma-Aldrich), were loaded into the wells and incubated for 45 minutesat 29° C. in 5% CO₂. Cells were then rinsed with either a 2 mM KClclosed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂,and 2 KCl, with the pH adjusted to 7.4 with NaOH) when performing theclosed-state assay or 12.5 mM KCl inactivated-state buffer (in mM: 128NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂, and 12.5 mM KCl, with the pHadjusted to 7.4 with NaOH) when performing the inactivated-state assay.

Concentration-dependent response curves were generated from 5 mM stocksolutions prepared in DMSO (Sigma-Aldrich) and diluted in either the 2mM KCl buffer or 12.5 mM KCl buffer and incubated for 20 minutes at 29°C. in 5% CO₂. Calcium entry was evoked with an addition of 130 mM KClstimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂,and 130 KCl, with the pH adjusted to 7.4 with NaOH) for both theclosed-state or inactivated-state assay. A change in the Fluo-4fluorescence signal was assessed using FLIPR^(TETRA)™ instrument(Molecular Devices, Sunnyvale, Calif.) for 3 minutes following theelevation of extracellular KCl using an illumination wavelength of470-495 nm with emissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing thefluorescence signal in the presence of compound and fitted with alogistic function (1) to obtain the concentration that inhibited 50%(IC₅₀) of the RLU signal using OriginPro v.7.5 software (OriginLab,Northampton, Mass.).

$\begin{matrix}{y = {\left\lbrack \frac{\max - \min}{1 + \left( \frac{\lbrack{drug}\rbrack}{{IC}_{50}} \right)^{n_{H}}} \right\rbrack + \min}} & (1)\end{matrix}$

To assess the quality of the FLIPR assays the Z-factor (2) were used toquantify the suitability of the assay conditions using the followingequation:

$\begin{matrix}{Z = {1 - \frac{{3{SD}_{sample}} + {3{SD}_{control}}}{{mean}_{sample} - {mean}_{control}}}} & (2)\end{matrix}$

Data were expressed as mean and standard deviation (SD).

C. High-Throughput Ca_(v)3.1 T-Type Fluorescent Assay

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysinecoated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior touse in the FLIPR assay. 100 μL of cells (2.0×10⁶ cell/mL) containingdoxycycline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression)were added to each well using a Multidrop (Thermo Scientific, Waltham,Mass.) and were maintained in 5% CO₂ incubator at 37° C. On the morningof the assay, cells were transferred to a 5% CO₂ incubator at 29° C.

Cells were washed with a wash buffer containing (in mM): 118 NaCl, 18.4HEPES, 11.7 D-glucose, 0.05 CaCl₂, 0.5 MgSO₄, 1 KCl, and 1.2 KH₂PO₄,with the pH adjusted to 7.2 with NaOH. 4.4 M of the fluorescentindicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid(Sigma-Aldrich), were loaded into the wells and incubated for 45 minutesat 29° C. in 5% CO₂. Cells were then rinsed with the following low Ca²buffer (in mM): 0.34 Na₂HPO₄, 4.2 NaHCO₃, 0.44 KH₂PO₄, 0.41 MgSO₄, 0.49MgCl₂-6H₂O, 20 HEPES, 5.5 D-Glucose, 137 NaCl, 5.3 KCl, and 0.001 CaCl₂,with 0.1% BSA and the pH adjusted to 7.2 with NaOH.Concentration-dependent response curves were generated from 5 mM stocksolutions prepared in DMSO (Sigma-Aldrich) and diluted in the buffercontaining low Ca² and incubated for 20 minutes at 29° C. in 5% CO₂Calcium entry was evoked with an addition of (in mM): 0.34 Na₂HPO₄, 4.2NaHCO₃, 0.44 KH₂PO₄, 0.41 MgSO₄. 0.49 MgCl₂-6H₂O, 20 HEPES, 5.5D-Glucose, 137 NaCl, 5.3 KCl, and 6 CaCl₂, with 0.1% BSA and the pHadjusted to 7.2 with NaOH. A change in the Fluo-4 fluorescence signalwas assessed using FLIPR^(TETRA)™ instrument (Molecular Devices,Sunnyvale, Calif.) for 3 minutes following the elevation ofextracellular KCl using an illumination wavelength of 470-495 nm withemissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing thefluorescence signal in the presence of compound and fitted with alogistic function (1) to obtain the concentration that inhibited 50%(IC₅₀) of the RLU signal using OriginPro v.7.5 software (OriginLab,Northampton, Mass.).

$\begin{matrix}{y = {\left\lbrack \frac{\max - \min}{1 + \left( \frac{\lbrack{drug}\rbrack}{{IC}_{50}} \right)^{n_{H}}} \right\rbrack + \min}} & (1)\end{matrix}$

To assess the quality of the FLIPR assays, the Z-factor (2) was used toquantify the suitability of the assay conditions using the followingequation:

$\begin{matrix}{Z = {1 - \frac{{3{SD}_{sample}} + {3{SD}_{control}}}{{mean}_{sample} - {mean}_{control}}}} & (2)\end{matrix}$

Data were expressed as mean and standard deviation (SD).

D. High-Throughput Ca_(v)3.2/K_(ir)2.3 T-Type Fluorescent Assay

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysinecoated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior touse in the FLIPR assay. 100 μL of cells (1.2×10⁶ cell/mL) containingdoxycycline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression)were added to each well using a Multidrop (Thermo Scientific, Waltham,Mass.) and were maintained in 5% CO₂ incubator at 37° C. On the morningof the assay, cells were transferred to a 5% CO₂ incubator at 29° C.

Cells were washed with a wash buffer containing (in mM): 118 NaCl, 18.4HEPES, 11.7 D-glucose, 2 CaCl₂, 0.5 MgSO₄, 4.7 KCl, and 1.2 KH₂PO₄, withthe pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicatordye Fluo-4 (Invitrogen) prepared in pluronic acid (Sigma-Aldrich) wereloaded into the wells and incubated for 45 minutes at 29° C. in 5% CO₂.Cells were then rinsed with either a 2 mM KCl closed-state buffer (inmM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂, and 2 KCl, with the pHadjusted to 7.4 with NaOH) when performing the closed-state assay or 7.6mM KCl inactivated-state buffer (in mM: 130.9 NaCl, 10 HEPES, 10D-glucose, 1 CaCl₂, and 7.6 mM KCl, with the pH adjusted to 7.4 withNaOH) when performing the inactivated-state assay.Concentration-dependent response curves were generated from 5 mM stocksolutions prepared in DMSO (Sigma-Aldrich), diluted in either the 2 mMKCl buffer or 7.6 mM KCl buffer, and incubated for 20 minutes at 29° C.in 5% CO₂. Calcium entry was evoked with an addition of either 12 mM KClstimulation buffer (in mM: 128.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂,and 12 KCl, with the pH adjusted to 7.4 with NaOH) or 14.5 mM KClstimulation buffer (in mM: 126 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂,and 14.5 KCl, with the pH adjusted to 7.4 with NaOH) for theclosed-state or inactivated-state assay respectively. A change in theFluo-4 fluorescence signal was assessed using FLIPR^(TETRA)™ instrument(Molecular Devices, Sunnyvale, Calif.) for 3 minutes following theelevation of extracellular KCl using an illumination wavelength of470-495 nm with emissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing thefluorescence signal in the presence of compound and fitted with alogistic function (1) to obtain the concentration that inhibited 50%(IC₅₀) of the RLU signal using OriginPro v.7.5 software (OriginLab,Northampton, Mass.).

$\begin{matrix}{y = {\left\lbrack \frac{\max - \min}{1 + \left( \frac{\lbrack{drug}\rbrack}{{IC}_{50}} \right)^{n_{H}}} \right\rbrack + \min}} & (1)\end{matrix}$

To assess the quality of the FLIPR assays, the Z-factor (2) was used toquantify the suitability of the assay conditions using the followingequation:

$\begin{matrix}{Z = {1 - \frac{{3{SD}_{sample}} + {3{SD}_{control}}}{{mean}_{sample} - {mean}_{control}}}} & (2)\end{matrix}$

Data were expressed as mean and standard deviation (SD).

E. High-Throughput Ca_(v)1.2/K_(ir)2.3 T-Type Fluorescent Assay

Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysinecoated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior touse in the FLIPR assay. 100 μL of cells (1.2×10⁶ cell/mL) containingdoxycycline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression)were added to each well using a Multidrop (Thermo Scientific, Waltham,Mass.) and were maintained in 5% CO₂ incubator at 37° C. On the morningof the assay, cells were transferred to a 5% CO₂ incubator at 29° C.

Cells were washed with a wash buffer containing (in mM): 118 NaCl, 18.4HEPES, 11.7 D-glucose, 2 CaCl₂, 0.5 MgSO₄, 4.7 KCl, and 1.2 KH₂PO₄, withthe pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicatordye Fluo-4 (Invitrogen) prepared in pluronic acid (Sigma-Aldrich) wereloaded into the wells and incubated for 45 minutes at 29° C. in 5% CO₂.Cells were then rinsed with either a 2 mM KCl closed-state buffer (inmM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂, and 2 KCl, with the pHadjusted to 7.4 with NaOH) when performing the closed-state assay or 30mM KCl inactivated-state buffer (in mM: 110.5 NaCl, 10 HEPES, 10D-glucose, 1 CaCl₂, and 30 mM KCl, with the pH adjusted to 7.4 withNaOH) when performing the inactivated-state assay.Concentration-dependent response curves were generated from 5 mM stocksolutions prepared in DMSO (Sigma-Aldrich), diluted in either the 2 mMKCl buffer or 30 mM KCl buffer, and incubated for 20 minutes at 29° C.in 5% CO₂. Calcium entry was evoked with an addition of 130 mM KClstimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl₂,and 130 KCl, with the pH adjusted to 7.4 with NaOH). A change in theFluo-4 fluorescence signal was assessed using FLIPR^(TETRA)™ instrument(Molecular Devices, Sunnyvale, Calif.) for 3 minutes following theelevation of extracellular KCl using an illumination wavelength of470-495 nm with emissions recorded at 515-575 nm.

Concentration-dependent response curves were obtained by comparing thefluorescence signal in the presence of compound and fitted with alogistic function (1) to obtain the concentration that inhibited 50%(IC₅₀) of the RLU signal using OriginPro v.7.5 software (OriginLab,Northampton, Mass.).

$\begin{matrix}{y = {\left\lbrack \frac{\max - \min}{1 + \left( \frac{\lbrack{drug}\rbrack}{{IC}_{50}} \right)^{n_{H}}} \right\rbrack + \min}} & (1)\end{matrix}$

To assess the quality of the FLIPR assays, the Z-factor (2) was used toquantify the suitability of the assay conditions using the followingequation:

$\begin{matrix}{Z = {1 - \frac{{3{SD}_{sample}} + {3{SD}_{control}}}{{mean}_{sample} - {mean}_{control}}}} & (2)\end{matrix}$

Data were expressed as mean and standard deviation (SD).

hERG K⁺ Channel Activity

It may be desirable that the compound has very low activity with respectto the hERG K⁺ channel, which is expressed in the heart: compounds thatblock this channel with high potency may cause reactions which arefatal. See, e.g., Bowlby et al., “hERG (KCNH2 or K_(v)11.1 K⁺ Channels:Screening for Cardiac Arrhythmia Risk.” Curr. Drug Metab. 9(9):965-70(2008)). Thus, for a compound that modulates, e.g., sodium channelactivity, it may also be shown that the hERG K⁺ channel is not inhibitedor only minimally inhibited as compared to the inhibition of the primarychannel targeted. Such compounds may be particularly useful in themethods described herein.

Compounds were tested using a standard electrophysiological assay (seeKiss et al., Assay & Drug Development Technologies, 1:1-2, 2003, andBridgland-Taylor et al., Journal of Pharmacological and ToxicologicalMethods, 54:189-199,2006). Briefly, compounds were tested at 3 μM usingIonWorks and the percent inhibition of the peak of the slowlydeactivating hERG tail current was used to assess the affinity.

Pain Models

L5/L6 Spinal Nerve Ligation (SNL)—Chung Pain Model

The Spinal Nerve Ligation is an animal model representing peripheralnerve injury generating a neuropathic pain syndrome. In this modelexperimental animals develop the clinical symptoms of tactile allodyniaand hyperalgesia. L5/L6 Spinal nerve ligation (SNL) injury was inducedusing the procedure of Kim and Chung (Kim et al., Pain 50:355-363(1992)) in male Sprague-Dawley rats (Harlan; Indianapolis, Ind.)weighing 200 to 250 grams. An exemplary protocol is provided below

The animals were anesthetized with isoflurane, the left L6 transverseprocess was removed, and the L5 and L6 spinal nerves were tightlyligated with 6-0 silk suture. The wound was closed with internal suturesand external tissue adhesive. Rats that exhibit motor deficiency (suchas paw-dragging) or failure to exhibit subsequent tactile allodynia canbe excluded from further testing.

Sham control rats can undergo the same operation and handling as theexperimental animals, but without SNL.

Assessment of Mechanical Hyperalgesia

Baseline and post-treatment values for mechanical hyperalgesia wereevaluated using a digital Randall-Selitto device (dRS; IITC LifeSciences, Woodland Hills, Calif.). Animals were allowed to acclimate tothe testing room for a minimum of 30 minutes before testing. Animalswere placed in a restraint sling that suspends the animal, leaving thehind limbs available for testing. Paw compression threshold was measuredonce at each time point for the ipsilateral and contralateral paws. Thestimulus was applied to the plantar surface of the hind paw by adome-shaped tip placed between the 3rd and 4th metatarsus, and pressurewas applied gradually over approximately 10 seconds. Measurements aretaken from the first observed nocifensive behavior of vocalization,struggle or withdrawal. A cut-off value of 300 g was used to preventinjury to the animal. The mean and standard error of the mean (SEM) weredetermined for each paw for each treatment group. Fourteen days aftersurgery, mechanical hyperalgesia was assessed and rats were assigned totreatment groups based on pre-treatment baseline values. Prior toinitiating drug delivery, baseline behavioural testing data can beobtained. At selected times after infusion of the Test or ControlArticle behavioural data can then be collected again.

Exemplary data are shown in FIGS. 2A-2C and 3A-3C for select compoundsof the invention. Additional data are presented in Tables 2-4 below.

Table 2 shows Compound (1) and Compound (41) (30 mg/kg, p.o.)significantly decreased mechanical hyperalgesia at 2 and 4 hours afteradministration compared to vehicle treated animals. Compound (24) (30mg/kg, p.o.) had no significant effect on mechanical hyperalgesia at anytime point tested compared to vehicle treated animals.

Table 3 shows that administration of Compound (33) (30 mg/kg, p.o.)significantly decreased mechanical hyperalgesia at 1, 2 and 4 hoursafter administration compared to vehicle treated animals. Administrationof Compound (56), Compound (31) or Compound (34) (30 mg/kg, p.o.)significantly decreased mechanical hyperalgesia 2 and 4 hours afteradministration compared to vehicle treated animals. Administration ofCompound (36) (30 mg/kg, p.o.) had no significant effect on mechanicalhyperalgesia at any time point tested compared to vehicle treatedanimals.

TABLE 2 Response Threshold (g) % Reversal % Gabapentin Compound 1 hr 2hr 4 hr 1 hr 2 hr 4 hr Peak Gabapentin 129.0 154.6 135.7 359. 55.3 41.0— (100 mg/kg; p.o.) DMA/PS80/PEG400 90.9 85.1 84.6 5.4 0.8 0.4 1.4(10:45:45, 2 mL/kg, p.o.) Compound (1) 93.8 120.6 113.3 9.2 30.2 24.454.7 (30 mg/kg, p.o.) Compound (24) 84.0 118.4 116.6 0.8 27.9 26.5 50.5(30 mg/kg, p.o.) Compound (41) 99.6 108.3 109.0 13.8 20.7 21.3 37.5 (30mg/kg, p.o.)

TABLE 3 Response Threshold (g) % Reversal % Gabapentin Compound 1 hr 2hr 4 hr 1 hr 2 hr 4 hr Peak Gabapentin 113.5 153.8 169.3 22.9 61.0 75.7— (100 mg/kg; p.o.) DMA/PS80/PEG400 87.8 85.5 83.2 −0.2 −2.2 −4.3 −3.7(10:45:45, 2 mL/kg, p.o.) Compound (31) 107.9 139.0 109.9 17.2 48.0 19.278.7 (30 mg/kg, p.o.) Compound (33) 113.6 136.9 150.8 22.4 44.7 58.173.3 (30 mg/kg, p.o.) Compound (34) 104.5 144.8 132.4 15.3 54.2 42.388.9 (30 mg/kg, p.o.) Compound (56) 110.8 131.4 140.4 19.8 40.4 49.466.2 (30 mg/kg, p.o.) Compound (36) 87.8 107.0 112.3 −0.5 18.6 23.8 30.5(30 mg/kg, p.o.)

Exemplary data are also shown in FIG. 2C and FIG. 3C for Compound (110)and in Table 4 below. Compound (110) is shown to significantly decreasemechanical hyperalgesia at two and four hours after administrationcompared to vehicle treated animals.

TABLE 4 Response Threshold (g) % Reversal % Gabapentin Compound 1 hr 2hr 4 hr 1 hr 2 hr 4 hr Peak Gabapentin 129.0 154.6 135.7 359. 55.3 41.0— (100 mg/kg; p.o.) DMA/PS80/PEG400 90.9 85.1 84.6 5.4 0.8 0.4 1.4(10:45:45, 2 mL/kg, p.o.) Compound (110) 100.6 148.5 126.1 14.6 51.234.1 92.7 (30 mg/kg, p.o.)

Assessment of Tactile Allodynia—Von Frey

The assessment of tactile allodynia can consist of measuring thewithdrawal threshold of the paw ipsilateral to the site of nerve injuryin response to probing with a series of calibrated von Frey filaments(innocuous stimuli). Animals can be acclimated to the suspendedwire-mesh cages for 30 min before testing. Each von Frey filament can beapplied perpendicularly to the plantar surface of the ligated paw ofrats for 5 sec. A positive response can be indicated by a sharpwithdrawal of the paw. For rats, the first testing filament is 4.31.Measurements can be taken before and after administration of testarticles. The paw withdrawal threshold can be determined by thenon-parametric method of Dixon (Dixon, Ann. Rev. Pharmacol. Toxicol.20:441-462 (1980)), in which the stimulus was incrementally increaseduntil a positive response was obtained, and then decreased until anegative result was observed. The protocol can be repeated until threechanges in behaviour were determined (“up and down” method; Chaplan etal., J. Neurosci. Methods 53:55-63 (1994)). The 50% paw withdrawalthreshold can be determined as (10^([Xf+kδ]))/10,000, where X_(f)=thevalue of the last von Frey filament employed, k=Dixon value for thepositive/negative pattern, and δ=the logarithmic difference betweenstimuli. The cut-off values for rats can be, for example, no less than0.2 g and no higher than 15 g (5.18 filament); for mice no less than0.03 g and no higher than 2.34 g (4.56 filament). A significant drop ofthe paw withdrawal threshold compared to the pre-treatment baseline isconsidered tactile allodynia. Rat SNL tactile allodynia can be testedfor the compounds described herein at, e.g., 60 minutes compared tobaseline and post-SNL.

Assessment of Thermal Hypersensitivity—Hargreaves

The method of Hargreaves and colleagues (Hargreaves et al., Pain 32:77-8(1988)) can be employed to assess paw-withdrawal latency to a noxiousthermal stimulus.

Rats may be allowed to acclimate within a Plexiglas enclosure on a clearglass plate for 30 minutes. A radiant heat source (e.g., halogen bulbcoupled to an infrared filter) can then be activated with a timer andfocused onto the plantar surface of the affected paw of treated rats.Paw-withdrawal latency can be determined by a photocell that halts bothlamp and timer when the paw is withdrawn. The latency to withdrawal ofthe paw from the radiant heat source can be determined prior to L5/L6SNL, 7-14 days after L5/L6 SNL but before drug, as well as after drugadministration. A maximal cut-off of 33 seconds is typically employed toprevent tissue damage. Paw withdrawal latency can be thus determined tothe nearest 0.1 second. A significant drop of the paw withdrawal latencyfrom the baseline indicates the status of thermal hyperalgesia.Antinociception is indicated by a reversal of thermal hyperalgesia tothe pre-treatment baseline or a significant (p<0.05) increase in pawwithdrawal latency above this baseline. Data is converted to % antihyperalgesia or % anti nociception by the formula: (100×(testlatency−baseline latency)/(cut-off−baseline latency) where cut-off is 21seconds for determining anti hyperalgesia and 40 seconds for determininganti nociception.

Epilepsy Models

6 Hz Psychomotor Seizure Model of Partial Epilepsy

Compounds can be evaluated for the protection against seizures inducedby a 6 Hz, 0.2 ms rectangular pulse width of 3 s duration, at a stimulusintensity of 32 mA (CC97) applied to the cornea of male CFI mice (20-30g) according to procedures described by Barton et al, “PharmacologicalCharacterization of the 6 Hz Psychomotor Seizure Model of PartialEpilepsy.” Epilepsy Res. 47(3):217-27 (2001). Seizures are characterisedby the expression of one or more of the following behaviours: stun,forelimb clonus, twitching of the vibrissae and Straub-tail immediatelyfollowing electrical stimulation. Animals can be considered “protected”if, following pre-treatment with a compound, the 6 Hz stimulus failed toevoke a behavioural response as describe above.

GAERS (Genetic Absence Epilepsy Rats from Strasbourg) Epilepsy Model

The GAERS (Genetic Absence Epilepsy Rats from Strasbourg) is noted forits long and frequently recurring absence seizure episodes.Investigators have determined, using electrophysiological recordingsfrom neurons within the thalamus, that the activity and expression ofthe low-voltage calcium channels is significantly increased in GAERS.Eight female GAERS rats, bred in the Ludwig Institute for CancerResearch, were used for this study. Rats weighed between 180 and 250 gand aged between 18 and 26 weeks at the start of the experiment. Methodsfor conducting this assay are known in the art.

Assessments of Neurological or Muscular Impairments

To assess a compound's undesirable side effects (toxicity), animals canbe monitored for overt signs of impaired neurological or muscularfunction. In mice, the rotarod procedure (Dunham et al., J. Am.Pharmacol. Assoc. 46:208-209 (1957)) is used to disclose minimalmuscular or neurological impairment (MMI). When a mouse is placed on arod that rotates at a speed of 6 rpm, the animal can maintain itsequilibrium for long periods of time. The animal is considered toxic ifit falls off this rotating rod three times during a 1-min period. Inaddition to MMI, animals may exhibit a circular or zigzag gait, abnormalbody posture and spread of the legs, tremors, hyperactivity, lack ofexploratory behavior, somnolence, stupor, catalepsy, loss of placingresponse and changes in muscle tone.

Recordings on Lamina I/II Spinal Cord Neurons

Male Wistar rats (P6 to P9 for voltage-clamp and P15 to P18 forcurrent-clamp recordings) can be anaesthetized through intraperitonealinjection of Inactin (Sigma). The spinal cord can then be rapidlydissected out and placed in an ice-cold solution protective sucrosesolution containing (in mM): 50 sucrose, 92 NaCl, 15 D-Glucose, 26NaHCO₃, 5 KCl, 1.25 NaH₂PO₄, 0.5 CaCl₂, 7 MgSO₄, 1 kynurenic acid, andbubbled with 5% CO₂/95% O₂. The meninges, dura, and dorsal and ventralroots can then removed from the lumbar region of the spinal cord under adissecting microscope. The “cleaned” lumbar region of the spinal cordmay be glued to the vibratome stage and immediately immersed in icecold, bubbled, sucrose solution. For current-clamp recordings, 300 to350 μm parasagittal slices can be cut to preserve the dendritic arbourof lamina I neurons, while 350 to 400 μm transverse slices can beprepared for voltage-clamped Na_(v) channel recordings. Slices may beallowed to recover for 1 hour at 35° C. in Ringer solution containing(in mM): 125 NaCl, 20 D-Glucose, 26 NaHCO₃, 3 KCl, 1.25 NaH₂PO₄, 2CaCl₂, 1 MgCl₂, 1 kynurenic acid, 0.1 picrotoxin, bubbled with 5%CO₂/95% O₂. The slice recovery chamber can then returned to roomtemperature (20 to 22° C.) for recordings.

Neurons may be visualized using IR-DIC optics (Zeiss Axioskop 2 FS plus,Gottingen, Germany), and neurons from lamina I and the outer layer oflamina II can be selected based on their location relative to thesubstantia gelatinosa layer. Neurons can be patch-clamped usingborosilicate glass patch pipettes with resistances of 3 to 6 MW.Current-clamp recordings of lamina I/II neurons in the intact slice, theexternal recording solution was the above Ringer solution, while theinternal patch pipette solution contained (in mM): 140 KGluconate, 4NaCl, 10 HEPES, 1 EGTA, 0.5 MgCl₂, 4 MgATP, 0.5 Na₂GTP, adjusted to pH7.2 with 5 M KOH and to 290 mOsm with D-Mannitol (if necessary). Tonicfiring neurons can be selected for current-clamp experiments, whilephasic, delayed onset and single spike neurons may be discarded (22).Recordings can be digitized at 50 kHz and low-pass filtered at 2.4 kHz.

Pharmacokinetic Parameters

Preliminary exposure characteristics of the compounds can be evaluatedusing, e.g., an in vivo Rat Early Pharmacokinetic (EPK) study design toshow bioavailability. For example, Male Sprague-Dawley rats can be dosedvia oral (PO) gavage in a particular formulation. Blood samples can thenbe collected from the animals at 6 timepoints out to 4 hours post-dose.Pharmacokinetic analysis can then performed on the LC-MS/MS measuredconcentrations for each timepoint of each compound.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

What is claimed is:
 1. A compound having a structure according to thefollowing formula,

or a pharmaceutically acceptable salt or solvate thereof, wherein eachof R¹, R², and R³ is, independently, H, unsubstituted C1-C6 alkyl,optionally substituted C1-C6 haloalkyl, or halogen; m is 1 or 2; each R⁴and R⁵ is, independently, H, optionally substituted C1-C6 alkyl, oroptionally substituted C1-C6 haloalkyl, or R⁴ and R⁵ combine to form anoptionally substituted C3-C6 cycloalkyl, or R⁴ and R⁵ combine to form anoxo (C═O) group; each of R⁶ and R⁸ is, independently, H or optionallysubstituted C1-C6 alkyl; or R⁶ and R⁸ combine to form an optionallysubstituted three-to-nine membered heterocyclyl, or R⁶ and R^(7A)combine to form an optionally substituted three-to-nine memberedheterocyclyl; n is 1 or 2; each R^(7A) and R^(7B) is, independently H,optionally substituted C1-C6 alkyl, or optionally substituted C1-C6haloalkyl; or R⁶ combines with R^(7A) to form an optionally substitutedthree-to-nine heterocyclyl; or an R^(7A) and R^(7B) group on the samecarbon combine to form an optionally substituted C3-C6 cycloalkyl; or,when n is 2, both R^(7A) groups combine to form an optionallysubstituted C3-C6 cycloalkyl.